RESUMEN
Cell disruption or lysis is a crucial step to obtain cellular components for various biological studies. We subjected different concentrations of Candida albicans to 5, 10, 15 and 20 cycles of disruption. The degree of cell lysis was observed using light microscopy and the yields obtained were measured and analysed. The optimum extraction with 1 x 10(10) yeast cells/ml was achieved after 5 cycles of disruption with 1.0 mm diameter glass beads at 5,000 rpm. Approximately 80% of the cells were lysed and the protein yield was 6,000 microg/ml. SDS-PAGE analysis revealed approximately 25 distinct protein bands with molecular weights ranging from 8 kDa to 220 kDa. We conclude that this mechanical disruption of fungal cells is a rapid, efficient and inexpensive technique for extracting whole cell proteins from yeast cells.
Asunto(s)
Candida albicans/química , Fraccionamiento Celular/métodos , Proteínas Fúngicas/aislamiento & purificación , Vibración , Candida albicans/citología , MalasiaRESUMEN
A range of cis- and trans-3-substituted 1-aminocyclobutane-1-carboxylic acids has been synthesized and evaluated for antagonism at excitatory amino acid receptor sites and for anticonvulsant activity. Potent and selective antagonist activity at N-methyl-D-aspartate (NMDA) receptor sites in neonatal rat motoneurones was shown by compounds in which the 3-substituent was, or contained, a 2'-carboxyethyl or 2'-phosphonoethyl moiety. Substances 4b, 24, 35, and 40 were more potent than the standard NMDA receptor antagonist, D-2-amino-5-phosphonopentanoate (D-AP5) as NMDA antagonists in this preparation, and about equipotent with [3-(+/-)-2-carboxypiperazin-4-yl)-1-propyl]phosphonate (CPP). Anticonvulsant activity, as assessed following intracerebroventricular injection into audiogenic DBA/2 mice, generally paralleled NMDA receptor antagonist activity.
Asunto(s)
Aminoácidos Cíclicos , Aminoácidos/química , Anticonvulsivantes/síntesis química , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Estimulación Acústica , Aminoácidos/farmacología , Aminoácidos/uso terapéutico , Animales , Animales Recién Nacidos , Anticonvulsivantes/uso terapéutico , Sitios de Unión , Simulación por Computador , Ratones , Ratones Endogámicos DBA , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Neuronas Motoras/metabolismo , Ratas , Convulsiones/etiología , Convulsiones/prevención & control , Médula Espinal/metabolismo , Relación Estructura-ActividadRESUMEN
1. The presynaptic depressant action of L-2-amino-4-phosphonobutyrate (L-AP4) on the monosynaptic excitation of neonatal rat motoneurones has been differentiated from the similar effects produced by (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate ((1S,3R)-ACPD), (1S,3S)-ACPD and (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG-I), and from the postsynaptic motoneuronal depolarization produced by (1S,3R)-ACPD, by the actions of two new antagonists, alpha-methyl-L-AP4 (MAP4) and alpha-methyl-L-CCG-I (MCCG). Such selectivity was not seen with a previously reported antagonist, (+)-alpha-methyl-4-carboxyphenylglycine (MCPG). 2. MAP4 selectively and competitively antagonized the depression of monosynaptic excitation produced by L-AP4 (KD 22 microM). At ten fold higher concentrations, MAP4 also antagonized synaptic depression produced by L-CCG-I but in an apparently non-competitive manner. MAP4 was virtually without effect on depression produced by (1S,3R)- or (1S,3S)-ACPD. 3. MCCG differentially antagonized the presynaptic depression produced by the range of agonists used. This antagonist had minimal effect on L-AP4-induced depression. The antagonism of the synaptic depression effected by (1S,3S)-ACPD and L-CCG-I was apparently competitive in each case but of varying effectiveness, with apparent KD values for the interaction between MCCG and the receptors activated by the two depressants calculated as 103 and 259 microM, respectively. MCCG also antagonized the presynaptic depression produced by (1S,3R)-ACPD. 4. Neither MAP4 nor MCCG (200-500 microM) significantly affected motoneuronal depolarizations produced by (1S,3R)-ACPD. At the same concentrations the two antagonists produced only very weak and variable effects (slight antagonism or potentiation) on depolarizations produced by (S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA).5. It is concluded that MAP4 is a potent and selective antagonist for those excitatory amino acid(EAA) receptors on neonatal rat primary afferent terminals that are preferentially activated by L-AP4,and that MCCG is a relatively selective antagonist for different presynaptic EAA receptors that are preferentially activated by (1S,3S)-ACPD and (perhaps less selectively) by L-CCG-I. These receptors probably comprise two sub-types of metabotropic glutamate receptors negatively linked to adenylyl cyclase activity.
Asunto(s)
Animales Recién Nacidos/fisiología , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Médula Espinal/efectos de los fármacos , Aminoácidos Dicarboxílicos/farmacología , Aminobutiratos/farmacología , Animales , Cicloleucina/análogos & derivados , Cicloleucina/farmacología , Aminoácidos Excitadores/farmacología , Técnicas In Vitro , Neuronas Motoras/efectos de los fármacos , N-Metilaspartato/farmacología , Neuronas Aferentes/efectos de los fármacos , Neurotoxinas/farmacología , Ratas , Sinapsis/efectos de los fármacosRESUMEN
The (+)-enantiomer of alpha-methyl-4-carboxyphenylglycine (MCPG) stereoslectively antagonized the depolarization of neonatal rat motoneurones and the excitation of rat thalamic neurons induced by the specific metabotropic glutamate receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (ACPD). (+)-MCPG preferentially reduced (1S,3R)-ACPD-induced responses relative to responses induced by (S)-alpha-amino-3-hydorxy-5-methylisoxazole-4-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA).
Asunto(s)
Animales Recién Nacidos/fisiología , Benzoatos/farmacología , Cicloleucina/análogos & derivados , Antagonistas de Aminoácidos Excitadores , Glicina/análogos & derivados , Neuronas Motoras/efectos de los fármacos , Neuronas/efectos de los fármacos , Neurotoxinas/antagonistas & inhibidores , Tálamo/citología , Animales , Cicloleucina/antagonistas & inhibidores , Cicloleucina/farmacología , Glicina/farmacología , Ácido Iboténico/análogos & derivados , Ácido Iboténico/farmacología , N-Metilaspartato/farmacología , Fármacos Neuromusculares Despolarizantes/farmacología , Neurotoxinas/farmacología , Ratas , Médula Espinal/citología , Médula Espinal/efectos de los fármacos , Estereoisomerismo , Tálamo/efectos de los fármacos , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol PropiónicoRESUMEN
The possible roles of G-protein coupled metabotropic glutamate receptors in central nervous function are currently the focus of intensive investigation. The complexity of effects produced by agonists at these receptors probably reflects the activity of a range of sub-types. The metabotropic glutamate receptors first described are linked to phospholipase C, mediating phosphoinositide hydrolysis and release of Ca2+ from intracellular stores. A substance generally considered to be a selective agonist for these receptors is (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD). This substance not only stimulates phosphoinositide hydrolysis, but also inhibits cyclic AMP formation. A family of metabotropic glutamate receptors, incorporating both phospholipase C- and adenylcyclase-linked sub-types has been cloned. Various effects of metabotropic glutamate receptor agonists on membrane ion fluxes and synaptic events have been reported, including neuronal depolarization and/or excitation, hyperpolarization, inhibition of Ca(2+)-dependent and voltage-gated K+ currents, potentiation of N-methyl-D-aspartate-induced responses, depression of synaptic excitation and either induction or augmentation of long-term potentiation. To clarify the role of metabotropic glutamate receptors in central nervous activity and to aid the characterization of the various receptor types that may be involved, a range of highly selective agonists and antagonists is required. To date, currently available antagonists such as L-2-amino-3-phosphonopropionate and L-aspartic acid-beta-hydroxamate appear to be unselective and insufficiently potent. We report here the actions of three phenylglycine derivatives, the particular agonist and/or antagonist properties of which may help to elucidate the roles of metabotropic glutamate receptors in central nervous activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Corteza Cerebral/fisiología , Cicloleucina/análogos & derivados , Glicina/análogos & derivados , Glicina/farmacología , Neuronas/fisiología , Receptores de Glutamato/fisiología , Médula Espinal/fisiología , Tálamo/fisiología , Animales , Animales Recién Nacidos , Corteza Cerebral/efectos de los fármacos , Cicloleucina/farmacología , Ácido Iboténico/análogos & derivados , Ácido Iboténico/farmacología , Técnicas In Vitro , Ácido Kaínico/farmacología , N-Metilaspartato/farmacología , Neuronas/efectos de los fármacos , Neurotoxinas/farmacología , Fosfatidilinositoles/metabolismo , Ratas , Receptores de Glutamato/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Tálamo/efectos de los fármacos , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol PropiónicoRESUMEN
Two phenylglycine derivates, (S)-4-carboxyphenylglycine and (RS)-alpha-methyl-4-carboxyphenylglycine, competitively antagonised (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (ACPD)-stimulated phosphoinositide hydrolysis in rat cerebral cortical slices. The same phenylglycine derivatives selectively antagonized ACPD-induced depolarization in neonatal rat spinal motoneurones and rate thalamic neurones relative to depolarization or excitation induced by N-methyl-D-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). Both phenylglycine derivatives also selectively depressed synaptic excitation in thalamic neurones evoked by noxious thermal stimuli, without affecting the synaptic stimulation of the same cells by non-noxious stimuli.
Asunto(s)
Benzoatos/farmacología , Antagonistas de Aminoácidos Excitadores , Glicina/análogos & derivados , Animales , Animales Recién Nacidos , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Cromatografía Líquida de Alta Presión , Cicloleucina/análogos & derivados , Cicloleucina/antagonistas & inhibidores , Cicloleucina/farmacología , Glicina/farmacología , Técnicas In Vitro , Neuronas Motoras/efectos de los fármacos , N-Metilaspartato/farmacología , Fosfatidilinositoles/metabolismo , Ratas , Médula Espinal/citología , Médula Espinal/efectos de los fármacos , Sinapsis/efectos de los fármacos , Tálamo/citología , Tálamo/efectos de los fármacosRESUMEN
Both the (1S,3S) and (1S,3R) stereoisomers of 1-aminocyclopentane-1,3-dicarboxylate (ACPD), but not the (1R,3R) or (1R,3S) isomers, potently depress the fastest (presumed monosynaptic) component of dorsal root-evoked ventral root potentials in hemisected isolated spinal cord of the newborn rat. This effect is not due to antagonism of known excitatory amino acid (EAA) receptors on motoneurones and may reflect an action of the two ACPD isomers at presynaptic EAA receptors of the L-2-amino-4-phosphonobutyrate (L-AP4) type.
Asunto(s)
Animales Recién Nacidos/fisiología , Cicloleucina/análogos & derivados , Neuronas Motoras/fisiología , Raíces Nerviosas Espinales/fisiología , Sinapsis/fisiología , Animales , Cicloleucina/química , Cicloleucina/farmacología , Isomerismo , Neuronas Motoras/efectos de los fármacos , Ratas , Sinapsis/efectos de los fármacosRESUMEN
1) The KD values for a range of antagonists including DGG, pCB-PzDA, pBB-PzDA, HDC-QXCA, DNQX and CNQX have been determined using a series of non-NMDA receptor agonists in the isolated spinal cord of the neonatal rat. 2) CNQX and DNQX, and, to a lesser extent, DCH-QXCA, were by far the most potent antagonists, although the degree of selectivity did not vary much throughout the whole range of antagonists used. 3) AMPA and domoate were the most and least sensitive agonists, respectively, to the action of all the antagonists. Ionophoretic experiments in the cat spinal cord in vivo confirmed this order of susceptibility in the case of the antagonists CNQX and pCB-PzDA. 4) Acromelic acid A was a more AMPA-like than domoate-like agonist. 5) The results suggest that two receptors contribute to the responses induced by several of the agonists, and that quisqualate and kainate are less selective agonists at these receptors than are AMPA and domoate, respectively.
Asunto(s)
Receptores de Neurotransmisores/clasificación , Animales , Técnicas In Vitro , Ácido Kaínico/antagonistas & inhibidores , Ácido Kaínico/metabolismo , Ácido Kaínico/farmacología , Ácido Quiscuálico/antagonistas & inhibidores , Ácido Quiscuálico/metabolismo , Ácido Quiscuálico/farmacología , Receptores AMPA , Receptores de Ácido Kaínico , Receptores de Neurotransmisores/efectos de los fármacos , Receptores de Neurotransmisores/metabolismo , Médula Espinal/metabolismoRESUMEN
The potency of 7 excitatory amino acid antagonists had been measured at kainate receptors on primary afferent C fibres using isolated dorsal roots from immature rats. Two of the compounds were tested as antagonists of excitant amino acids at motoneurones using isolated hemisected spinal cord preparations. Mean dose-ratios for antagonism of kainate at C fibres, produced by 1 mM antagonist in at least four preparations, ranged from 2.1 +/- 0.5 (mean +/- s.e.mean) with 2-amino-5-phosphonopentanoate (AP5) to 17.6 +/- 2.4 with 1-(p-bromobenzoyl)-piperazine-2, 3-dicarboxylate (BBpzD). The rank order of potency of antagonists at C fibres was similar to that obtained previously for antagonism of kainate at motoneurones. The potency of kynurenate as an antagonist of kainate at C fibres (apparent Kd 70 +/- 4.3 microM (mean +/- s.e.mean), n = 12) was significantly different (P less than 0.005, Wilcoxon rank sum) from its potency at motoneurones (apparent Kd 164 +/- 14 microM, n = 13). Kynurenate also was significantly (P less than 0.025 Wilcoxon rank sum) more potent at antagonizing kainate- than quisqualate (apparent Kd 258 +/- 28 microM, n = 12)-induced depolarization of motoneurones. Kynurenate and BBpzD (0.25, 1.0 and 4.0 mM) were compared as antagonists of N-methyl-D-aspartate (NMDA) at motoneurones and the slope of the Gaddum-Schild plot for kynurenate was markedly greater than 1 (2.01 +/- 0.22, 95% confidence limits). A greater than additive antagonism of NMDA-induced depolarizations was produced by combinations of kynurenate with, either AP5, or magnesium ions. 5. The results suggest that the kainate receptor on C fibres may be different from the kainate receptor on motoneurones and that antagonism of NMDA-induced depolarization by kynurenate may not be entirely competitive.