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Is the heterologous expression of metabotropic glutamate receptors (mGluRs) an appropriate method to study the mGluR function? Experience with human embryonic kidney 293 cells transfected with mGluR1.
No full textThe cloning of metabotropic glutamate receptors (mgluRs) has initiated a new approach to the study of their function: the introduction of mGluR cDNA into cells that do not normally express mGluRs, thus allowing the heterologous receptor expression. We have transfected human embryonic kidney (HEK) 293 cells with the full length mGluR1a cDNA and with its truncated variant which encodes the receptor termed mGluR1T (a receptor lacking the long intracellular domain and similar to the splice variant mGluR1c). Transient transfection of HEK-293 cells with mGluR1a, but not the mGluR1T cDNA, resulted in a significant increase in inositol phosphate (IP) formation in absence of any mGluR agonists. This effect was completely dependent on the presence of extracellular calcium, and unlike the agonist-stimulated IP formation it was insensitive to pertussis toxin. The prolonged activation of IP formation might affect the cell physiology. In an attempt to obtain stably transfected cells, we transfected about 1.5 x 10(6) HEK-293 cells with the plasmid conveying the full-length mGluR1a cDNA and the neomycin-resistance gene. Only 12 clones survived the antibiotic selection, and only one of these 12 clones continued to divide. The size of mRNA from the clone was smaller than the full-length mGluR1a mRNA. The shortened mRNA, revealed in the clone, apparently encoded a functional mGluR that was sensitive to glutamate, but unlike the mGluR1a, it did not respond to 1S,3R-ACPD (1S,3R-aminocyclopentane-1,3-dicarboxylic acid). A prudent use of the heterologous cell transfection technique is necessary in studying the function and the pharmacology of mGluRs
Polyamines modulate the function of transfected glutamate receptor mGluR1a.
No full textMetabotropic glutamate receptor mGluR1a was expressed in human embryonic kidney 293 cells. 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) stimulated dose-dependently, phosphoinositide (PI) hydrolysis in transfected, but not in non-transfected cells. The polyamine spermine did not affect PI hydrolysis in the absence of 1S,3R-ACPD even at a concentration of 1 mM, but it potentiated the stimulatory action of 1S,3R-ACPD at 10 microM. The modulatory action of spermine was mimicked by spermidine but not by the short polyamine putrescine
DIFFERENT TRANSDUCTION SYSTEMS ARE ASSOCIATED WITH METABOTROPIC GLUTAMATE-RECEPTOR MGLUR1 DEPENDING ON THE LENGTH OF ITS CARBOXYL-TERMINAL DOMAIN
No full textHeterologous expression of full-length metabotropic glutamate receptor and of a truncated isoform lacking the C-terminal domain was perfomed. Characterization of the actity and of the receptor coulpling to singal transduction pathways revealed that different G-proteins are coupled with the two isoforms
Functional evidence for a L-AP3-sensitive metabotropic receptor different from glutamate metabotropic receptor mGluR1.
No full textThe efficacy of mGluR agonists quisqualate and 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) in stimulating the inositol phosphate (IP) formation in primary cultures of cerebellar granule neurons correlated with mGluR1 mRNA expression and was affected by the medium KCl content. L-2-Amino-3-phosphonopropionic acid (L-AP3) mimicked the stimulatory action of mGluR agonists. Maximal stimulatory doses of mGluR agonist 1S,3R-ACPD and L-AP3 were additive, suggesting the action of L-AP3 on a receptor different from mGluR1. Indeed, in embryonic kidney 293 cells transfected with mGluR1 cDNA quisqualate and 1S,3R-ACPD but not L-AP3 stimulated the IP formation
Carboxyl domain of glutamate receptor directs its coupling to metabolic pathways.
No full textOf the six metabotropic glutamate receptors (mGluRs) only mGluR1 and mGluR5, which possess a large carboxyl terminal domain, are positively linked to phosphoinositide (PI) hydrolysis. We expressed a 3' deletion of mGluR1 alpha (mGluR1T) lacking the terminal 290 codons and the full length mGluR1 alpha cDNAs in human embryonic kidney 293 cells. Agonist stimulation of both mGluR1 alpha and mGluR1T stimulated PI hydrolysis. Glutamate activation of PI hydrolysis was reduced by pertussis toxin when mediated via mGluR1 alpha, while mGluR1T required the presence of extracellular Ca2+. Glutamate-mediated reduction of adenylyl cyclase stimulation by forskolin occurred only in mGluR1T-expressing cells. The results suggest that the carboxyl terminal extension directs the coupling of mGluR1 with different signal transduction pathways
Trans-azetidine-2,4-dicarboxylic acid activates neuronal metabotropic receptors.
No full textThe expression of metabotropic glutamate receptors (mGluRs) in primary cultures of cerebellar granule neurones can be: (i) modulated by the degree of depolarization during the culture period, rendering neurones differently sensitive to agonist-stimulated inositol phosphate (IP) hydrolysis; (ii) down-regulated by specific mGluR agonists. In this culture the new rigid glutamate analogue, (+/-)-trans-azetidine-2,4-dicarboxylic acid (t-ADA) and the known mGluR agonist 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) stimulated IP formation in line with the depolarization-modified expression of mGluR1. However, the two compounds caused different patterns of mGluR down-regulation. The effects of t-ADA and 1S,3R-ACPD were also tested on transformed human embryonic kidney 293 cells transfected with mGluR1. Only 1S,3R-ACPD, but not t-ADA, stimulated IP hydrolysis, suggesting that t-ADA acts on a subtype of metabotropic receptors different from mGluR1. Hence, t-ADA might prove useful in differentiating the function of various mGluR subtypes
PHOTOCHEMICAL STROKE AND BRAIN-DERIVED NEUROTROPHIC FACTOR (BDNF) MESSENGER-RNA EXPRESSION
No full textIn situ hybridization and Northern blotting were used to study the expression of brain-derived neurotrophic factor (BDNF) mRNA in the rat brain following photochemical stroke. A focal thrombotic lesion of the sensorimotor cortex was produced by intravenously injecting the light-sensitive dye rose bengal and exposing the skull to a controlled beam of light. Four hours after the light exposure the level of BDNF mRNA was increased in the hippocampus and cortex ipsilateral and perifocal to the lesion. The stroke-induced BDNF mRNA increase was prevented by the non-competitive glutamate receptor blocker dizocilpine (MK-801). The results indicate that the activation of N-methyl-D-aspartate (NMDA)-sensitive glutamate receptors is involved in the stroke-triggered stimulation of BDNF mRNA increase
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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