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1.
Abstract : We have isolated and characterized overlapping cDNAs encoding a novel, voltage-gated Ca2+ channel α1 subunit, α1H, from a human medullary thyroid carcinoma cell line. The α1H subunit is structurally similar to previously described α1 subunits. Northern blot analysis indicates that α1H mRNA is expressed throughout the brain, primarily in the amygdala, caudate nucleus, and putamen, as well as in several nonneuronal tissues, with relatively high levels in the liver, kidney, and heart. Ba2+ currents recorded from human embryonic kidney 293 cells transiently expressing α1H activated at relatively hyperpolarized potentials (-50 mV), rapidly inactivated (τ = 17 ms), and slowly deactivated. Similar results were observed in Xenopus oocytes expressing α1H. Singlechannel measurements in human embryonic kidney 293 cells revealed a single-channel conductance of ~9 pS. These channels are blocked by Ni2+ (IC50 = 6.6 μ M ) and the T-type channel antagonists mibefradil (~50% block at 1 μ M ) and amiloride (IC50 = 167 μ M ). Thus, α1H-containing channels exhibit biophysical and pharmacological properties characteristic of low voltage-activated, or T-type, Ca2+ channels.  相似文献   

2.
The problem of whether a given amount of active ingredient is more effectively applied in concentrated or dilute form is discussed. If y is percentage mortality and x 1 and x 2 are respectively log-concentration of active ingredient and log-volume (or deposit) of insecticide applied, y may be regarded as a uniform, continuous function of x 1 and x 2. The amount of active ingredient is constant, i.e. x 1+ x 2= k , so that
dx/dx1=−dy/dx2=δy/δx1−δy/δx2
Probit mortality, Y , can be substituted for y in (4). Thus, whether an active ingredient is better applied in concentrated or dilute form depends on the relative magnitudes of ∂ y /∂ x 1 and ∂ y /∂ x 2, or of ∂ Y /∂ x 1 and ∂ Y /∂ x 2. Equation (4) is true whenever an insecticide consists of an active ingredient in a diluent, whatever the dosage-mortality relationship. Previous work is discussed in the light of (4) and its probit form, and it appears that the concentration at which an active ingredient is best applied can depend upon the nature and quantity of the active ingredient, and the method of application of the spray. There may be other facto*** The probit form of (4) is applied to the probit plane and confirmed experimentally. Flour beetles, Tribolium castaneum Herbst were sprayed with pyrethrins in Shell oil P 31, and it was found that ∂ Y /∂ x 1 > ∂ Y /∂ x 2, so that a given quantity of pyrethrins was more toxic in concentrated solution than in dilute.  相似文献   

3.
SUMMARY. Information on temperature (T°C) and time from fertilization to 50% hatch ( D days) for five species of salmonid fishes has been used to assess several mathematical models relating D and T . No single equation gave the best fit to all five data sets. The power law with temperature correction (α), log101 D = log10 a + b log10 ( T - α) and the quadratic, log10 D = log10 a + bT + b 1 T 2 (where a, b, b 1, and α are constants), each accounted for over 97 % of the variance of D and were good fits to the observed data points for all five species. There was little difference between the predictions obtained from these two equations within the range of observed temperatures. Therefore, the simpler power-law model is preferred. However, there were substantial within-species differences between values of D predicted from extrapolations of the two models from 2 or 3°C down to 0°C. When more data for low temperatures become available it will be possible to make a more objective choice of model.  相似文献   

4.
5.
Zolpidem is a positive allosteric modulator of GABAA receptors with sensitivity to subunit composition. While it acts with high affinity and efficacy at GABAA receptors containing the α1 subunit, it has a lower affinity to GABAA receptors containing α2, α3, or α5 subunits and has a very weak efficacy at receptors containing the α5 subunit. Here, we show that replacing histidine in position 105 in the α5 subunit by cysteine strongly stimulates the effect of zolpidem in receptors containing the α5 subunit. The side chain volume of the amino acid residue in this position does not correlate with the modulation by zolpidem. Interestingly, serine is not able to promote the potentiation by zolpidem. The homologous residues to α5H105 in α1, α2, and α3 are well-known determinants of the action of classical benzodiazepines. Other studies have shown that replacement of these histidines α1H101, α2H101, and α3H126 by arginine, as naturally present in α4 and α6, leads to benzodiazepine insensitivity of these receptors. Thus, the nature of the amino acid residue in this position is not only crucial for the action of classical benzodiazepines but in α5 containing receptors also for the action of zolpidem.  相似文献   

6.
7.
Abstract: Cerebral cortical [3H]clonidine binding and influences of GTP and cations were investigated in developing rats. The results from Scatchard plots were compatible with the presence of two populations of binding sites [high-affinity binding ( KD = 0.59 n M ) and low-affinity binding ( KD = 7.12 n M )] in 70-day-old rats but only high-affinity binding ( KD = 0.27 n M ) on day 1. Low-affinity binding was detectable on day 7. KD values in high- and low-affinity binding were not significantly changed during development after 7 days. Bmax of high-affinity binding reached a peak on day 15, and the value of low-affinity binding gradually increased with age. The addition of 10 μ M GTP caused a significant reduction in Bmax of high-affinity binding after day 7. Neither K D nor Bmax of low-affinity binding was affected by 10 μ M GTP during development. NaCl (10 and 100 m M ) diminished the binding on days 7 and 70. MnCl2 (0.1 and 1.0 m M ) markedly increased the binding on days 15 and 70 but not on day 7. It is suggested that: (1) single binding sites of α2-adrenoceptors with higher affinity seem to be present on day 1; (2) low-affinity binding appears on day 7; (3) the number of high-affinity binding sites reaches a peak on day 15, followed by changes in populations of high-affinity as well as low-affinity sites without changing affinity; (4) the regulatory mechanism in α2-receptors by guanine nucleotide reaches functional maturity between days 1 and 7; and (5) the involvement of Na+ and Mn2+ in α2-receptor binding becomes functional by days 7 and 15, respectively.  相似文献   

8.
Abstract: Progesterone and its A-ring reduced metabolites are allosteric activators of GABAA receptors. The studies reported here examined the effects of these steroids on brain nicotinic receptors using an 86Rb+ efflux assay that likely measures the function of α4β2-type nicotinic receptors and [3H]dopamine release, which may be modulated by an α3-containing nicotinic receptor. Both of the A-ring reduced metabolites of progesterone were noncompetitive inhibitors of both assays, whereas progesterone inhibited only the 86Rb+ efflux assay. The 86Rb+ efflux assay was slightly more sensitive than was the dopamine release assay to steroid inhibition. Inhibition developed slowly for both assays ( t 1/2 = 0.4 min) and was reversed even more slowly ( t 1/2 = 10–15 min). Steroid addition did not alter either the rate of association of [3H]nicotine binding to brain membranes, nor was equilibrium binding changed. These findings argue that neurosteroids are allosteric inhibitors of brain nicotinic receptors.  相似文献   

9.
Abstract: The interactions of the atypical benzodiazepine 4'-chlorodiazepam (Ro 5-4864) with functionally expressed human GABAA receptor cDNAs were determined. Cotransfection of human α2, β1, and γ2 subunits was capable of reconstituting a 4'-chlorodiazepam recognition site as revealed by a dose-dependent potentiation of t -[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to the GABA-activated chloride channel. This site is found on GABAA receptor complexes containing sites for GABA agonist-like benzodiazepines and neuroactive steroids. The importance of the α subunit was further demonstrated as substitution of either α1 or α3 for the α2 subunit did not reconstitute a 4'-chlorodiazepam recognition site that was capable of modulating [35S]TBPS binding under the same experimental conditions. The 4'-chlorodiazepam modulatory site was shown to be distinct from the benzodiazepine site, but the phenylquinolines PK 8165 and PK 9084 produced effects similar to 4'-chlorodiazepam, consistent with the previous analysis of the 4'-chlorodiazepam site in brain homogenates. Further analysis of the subunit requirements revealed that coexpression of α2 and β1 alone reconstituted a 4'-chlorodiazepam recognition site. It is interesting, however, that the 4'-chlorodiazepam site was found to inhibit [35S]TBPS binding to the GABA-activated chloride channel. Thus, the 4'-chlorodiazepam site may be reconstituted with only the α and β polypeptides.  相似文献   

10.
Calcium/calmodulin-dependent kinase II (CaMKII) facilitates L-type calcium channel (LTCC) activity physiologically, but may exacerbate LTCC-dependent pathophysiology. We previously showed that CaMKII forms stable complexes with voltage-gated calcium channel (VGCC) β1b or β2a subunits, but not with the β3 or β4 subunits ( Grueter et al. 2008 ). CaMKII-dependent facilitation of CaV1.2 LTCCs requires Thr498 phosphorylation in the β2a subunit ( Grueter et al. 2006 ), but the relationship of this modulation to CaMKII interactions with LTCC subunits is unknown. Here we show that CaMKII co-immunoprecipitates with forebrain LTCCs that contain CaV1.2α1 and β1 or β2 subunits, but is not detected in LTCC complexes containing β4 subunits. CaMKIIα can be specifically tethered to the I/II linker of CaV1.2 α1 subunits in vitro by the β1b or β2a subunits. Efficient targeting of CaMKIIα to the full-length CaV1.2α1 subunit in transfected HEK293 cells requires CaMKII binding to the β2a subunit. Moreover, disruption of CaMKII binding substantially reduced phosphorylation of β2a at Thr498 within the LTCC complex, without altering overall phosphorylation of CaV1.2α1 and β subunits. These findings demonstrate a biochemical mechanism underlying LTCC facilitation by CaMKII.  相似文献   

11.
Abstract: The excitatory amino acid glutamate was previously shown to stimulate aerobic glycolysis in astrocytes by a mechanism involving its uptake through an Na+-dependent transporter. Evidence had been provided that Na+,K+-ATPase might be involved in this process. We have now measured the activity of Na+,K+-ATPase in cultured astrocytes, using ouabain-sensitive 86Rb uptake as an index. l -Glutamate increases glial Na+,K+-ATPase activity in a concentration-dependent manner with an EC50 = 67 µ M . Both l - and d -aspartate, but not d -glutamate, produce a similar response, an observation that is consistent with an uptake-related effect rather than a receptor-mediated one. Under basal conditions, concentration-dependent inhibition of Na+,K+-ATPase activity in astrocytes by ouabain indicates the presence of a single catalytic site with a low affinity for ouabain ( K 0.5 = 113 µ M ), compatible with the presence of an α1 isozyme. On stimulation with glutamate, however, most of the increased activity is inhibited by low concentrations of ouabain ( K 0.5 = 20 n M ), thus revealing a high-affinity site akin to the α2 isozyme. These results suggest that astrocytes possess a glutamate-sensitive isoform of Na+,K+-ATPase that can be mobilized in response to increased neuronal activity.  相似文献   

12.
Abstract: The pentameric subunit composition of a large population (36%) of the cerebellar granule cell GABAA receptors that show diazepam (or clonazepam)-insensitive [3H]Ro 15-4513 binding has been determined by immunoprecipitation with subunit-specific antibodies. These receptors have α6, α1, γ2S, γ2L, and β2 or β3 subunits colocalizing in the same receptor complex.  相似文献   

13.
Abstract: Using receptors expressed from mouse brain mRNA in Xenopus oocytes, we found that enhancement of type A γ-aminobutyric acid (GABAA) receptor-gated Cl channel response is a common action of structurally diverse anesthetics, suggesting that the GABAA receptor plays an important role in anesthesia. To determine if GABAA receptor subunit composition influences actions of anesthetics, we expressed subunit cRNAs in Xenopus oocytes and measured effects of enflurane on GABA-activated Cl currents. Potentiation of GABA-activated currents by enflurane was dependent on the composition of GABAA receptor protein subunits; the order of sensitivity was α1β1 > α1β1γ2s1β1γ2L > total mRNA. The results suggest that anesthetics with simple structures may act on the GABAA receptor protein complex to modulate the Cl channel activity and provide a molecular explanation for the synergistic clinical interactions between benzodiazepines and general anesthetics.  相似文献   

14.
Abstract: Polyclonal antibodies were raised to synthetic peptides having amino acid sequences corresponding with the N- or C-terminal part of the γ-aminobutyric acidA (GABAA) receptor α5-subunit. These anti-peptide α5(2–10) or anti-peptide α5(427–433) antibodies reacted specifically with GABAA receptors purified from the brains of 5–10-day-old rats in an enzyme-linked immunosorbent assay and were able to dose-dependently immunoprecipitate up to 6.3 or 13.1% of the GABAA receptors present in the incubation, respectively. In immunoblots, each of these antibodies reacted with the same two protein bands with apparent molecular mass of 53 or 57 kDa. After exhaustive treatment of purified GABAA receptors with N -Glycanase, each of these antibodies identified two proteins with apparent molecular masses of 46 and 48 kDa. Additional treatment of GABAA receptors with neuraminidase and O -Glycanase resulted in an apparently single protein with molecular mass of 47 kDa, which again was identified by both the anti-peptide α5(2–10) and the anti-peptide α5(427–433) antibody. These results indicate the existence of at least two different α5-sub-units of the GABAA receptor that differ in their carbohydrate content. In contrast to other α- or β-subunits of GABAA receptors so far investigated, at least one of these two α5-subunits contains O-linked carbohydrates.  相似文献   

15.
Abstract: This study examines the proteolysis of the carboxy terminal domain of the full-length (α1212) and truncated (α1190) forms of the rabbit skeletal muscle L-type calcium channel α1 subunit by calpain I and calpain II. Although both forms of the α1 subunit show little sensitivity to proteolysis by calpain II, α1212 is relatively more sensitive than α1190 to digestion by calpain I, the form of the enzyme regulated by micromolar concentrations of calcium. Calpain I cleaves a 37-kDa fragment from the C-terminus of α1212 in a time- and concentration-dependent manner and proteolysis is independent of the α1212 phosphorylation state. This proteolytic cleavage removes the major site of cyclic AMP-dependent phosphorylation from α1212 and may provide a mechanism for modifying the cyclic AMP-dependent regulation of L-type calcium channels in skeletal muscle.  相似文献   

16.
Abstract: Aromatase in the diencephalic neurons, the level of which increases transiently during the prenatal to neonatal period, has been suggested to be involved in control of sexual behavior and differentiation of the CNS. Effects of neurotransmitters on levels of aromatase mRNA in cultured neurons were investigated to determine factors regulating the developmental increase that occurs in level of fetal brain aromatase. The expression of aromatase in diencephalic neurons of fetal mice at embryonic day 13, cultured in vitro, was significantly affected by α1-adrenergic receptor ligands. Aromatase mRNA levels were higher in neurons treated with the α1-agonist phenylephrine than in control neurons, whereas prazosin, an α1-antagonist, suppressed this increase, and ligands for α2- or β-adrenergic receptors did not exert any influence. The profile of α1-adrenergic receptor subtypes during actual development in vivo suggested that the α1B subtype is in fact responsible for the signal transduction. Substance P, cholecystokinin, neurotensin, and brain natriuretic peptide also increased the level of expression along with phorbol 12-myristate 13-acetate and dibutyryl-cyclic GMP, whereas forskolin and dibutyryl-cyclic AMP caused a decrease. These data indicate that stimulation via α1 (possibly α1B)-adrenergic receptors, as well as receptors of specific neuropeptides, controls the expression of aromatase in embryonic day 13 diencephalic neurons through activation of protein kinase C or G. β-Adrenergic receptors would not appear to participate in the regulation, judging from their developmental profile, although cyclic AMP might be a suppressive second messenger.  相似文献   

17.
The molecular pathogenesis of infections caused by group A Streptococcus (GAS) is not fully understood. We recently reported that a recombinant protein derived from the collagen-like surface protein, Scl1, bound to the human collagen receptor, integrin α2β1. Here, we investigate whether the same Scl1 variant expressed by GAS cells interacts with the integrin α2β1 and affects the biological outcome of host–pathogen interactions. We demonstrate that GAS adherence and internalization involve direct interactions between surface expressed Scl1 and the α2β1 integrin, because (i) both adherence and internalization of the scl1- inactivated mutant were significantly decreased, and were restored by in-trans complementation of Scl1 expression, (ii) GAS internalization was reduced by pre-treatment of HEp-2 cells with anti-α2 integrin-subunit antibody and type I collagen, (iii) recombinant α2-I domain bound the wild-type GAS cells and (iv) internalization of wild-type cells was significantly increased in C2C12 cells expressing the α2β1 integrin as the only collagen-binding integrin. Next, we determined that internalized GAS re-emerges from epithelial cells into the extracellular environment. Taken together, our data describe a new molecular mechanism used by GAS involving the direct interaction between Scl1 and integrins, which increases the overall capability of the pathogen to survive and re-emerge.  相似文献   

18.
Abstract: Tolerance to and withdrawal from pentobarbital were induced in rats by continuous intracerebroventricular infusion via subcutaneously implanted osmotic minipumps. In situ hybridization of GABAA receptor α1- and β3-subunit mRNA was conducted using synthetic 3'- end 35S-dATP-labeled oligodeoxynucleotide probes. Results were quantified by film densitometry. In animals that were tolerant to pentobarbital, levels of α1-subunit mRNA were decreased in hippocampus, superior colliculus, and inferior colliculus, but levels of β3-subunit mRNA were not affected. Dramatically increased levels of GABAA receptor subunit mRNA were observed in animals 24 h after withdrawal from chronic pentobarbital treatment. These increases occurred in cerebral cortex and cerebellum for the α1 subunit and in cerebral cortex only for the β3-subunit. These data provide further support to the structural and pharmacological GABAA receptor heterogeneity in discrete brain areas. The observed changes of subunit expression may underlie, at least in part, the receptor up- and down-regulation observed in receptor ligand binding studies.  相似文献   

19.
Abstract: A novel fluorescent Na+ indicator, Na+-binding benzofuran isophthalate (SBFI), was used to follow changes in the intracellular free Na+ concentration ([Na+]1) of synaptosomes. The dye, when loaded into synapto- somes in the form of its acetoxymethyl ester, was responsive to changes of [Na+]1. Calibration was made using the 340/380 nm excitation ratio when the cytoplasmic Na+ concentration was equilibrated with different concentrations of extracellular Na+ in the presence of 2 μ M gramicidin D. The basal value of [Na+]1 in synaptosomes in the presence of 140 m M extracellular Na+ was found to be 10.9 ± 1.8 m M. Veratridine, which opens potential-dependent Na+ channels, caused a sudden increase in [Na+]1 in a concentration-dependent manner (1 -20 μ M ), whereas the effect of ouabain (20 and 50 μ M ), the inhibitor of the plasma membrane Na+,K+-ATPase, was more gradual. The rise in the fluorescence intensity upon addition of veratridine was prevented completely by 2 μ M tetrodotoxin. α-Latrotoxin, the black widow spider toxin, caused an increase in the fluorescence intensity, which became evident 1 min after the addition of the toxin. The rate of increase was proportional to the concentration of the toxin (0.19–1.5 n M ). This report confirms our earlier finding demonstrating a Na+-dependent component in the action of α-Iatrotoxin, and shows that changes in [Na+]1 in synaptosomes can be followed by SBFI.  相似文献   

20.
Five strains of filamentous fungi belonging to the genera Mortierella and Cunninghamella were examined for the content of dihomo-γ-linolenic, arachidonic, eicosapentaenoic acids and prostaglandins (type E2 and F ). Prostaglandins were detected using an ELISA method in mycelia of all tested strains (range 50–4800 ng g−1 of PGE2 and 6–30 ng g−1 of PG F ). Several micro-organisms also produced prostaglandins in the culture medium (2·2–137·6 μg l−1 for PGE2 and 0·4–7·8 μg l−1 for PG F ).  相似文献   

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