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1.
Killing of wild-type spores of Bacillus subtilis with formaldehyde also caused significant mutagenesis; spores (termed αβ) lacking the two major α/β-type small, acid-soluble spore proteins (SASP) were more sensitive to both formaldehyde killing and mutagenesis. A recA mutation sensitized both wild-type and αβ spores to formaldehyde treatment, which caused significant expression of a recA - lacZ fusion when the treated spores germinated. Formaldehyde also caused protein–DNA cross-linking in both wild-type and αβ spores. These results indicate that: (i) formaldehyde kills B. subtilis spores at least in part by DNA damage and (b) α/β-type SASP protect against spore killing by formaldehyde, presumably by protecting spore DNA.  相似文献   

2.
GABAA receptors are pentameric ligand-gated ion channels that are major mediators of fast inhibitory neurotransmission. Clinically relevant GABAA receptor subtypes are assembled from α5(1-3, 5), β1-3 and the γ2 subunit. They exhibit a stoichiometry of two α, two β and one γ subunit, with two GABA binding sites located at the α/β and one benzodiazepine binding site located at the α/γ subunit interface. Introduction of the H105R point mutation into the α5 subunit, to render α5 subunit-containing receptors insensitive to the clinically important benzodiazepine site agonist diazepam, unexpectedly resulted in a reduced level of α5 subunit protein in α5(H105R) mice. In this study, we show that the α5(H105R) mutation did not affect cell surface expression and targeting of the receptors or their assembly into macromolecular receptor complexes but resulted in a severe reduction of α5-selective ligand binding. Immunoprecipitation studies suggest that the diminished α5-selective binding is presumably due to a repositioning of the α5(H105R) subunit in GABAA receptor complexes containing two different α subunits. These findings imply an important role of histidine 105 in determining the position of the α5 subunit within the receptor complex by determining the affinity for assembly with the γ2 subunit.  相似文献   

3.
The H+/PPi stoichiometry of the mitochondrial H+‐PPiase from pea ( Pisum sativum L.) stem was determined by two kinetic approaches, and compared with the H+/substrate stoichiometries of the mitochondrial H+‐ATPase, and the vacuolar H+‐PPiase and H+‐ATPase. Using sub‐mitochondrial particles or preparations enriched in vacuolar membranes, the rates of substrate‐dependent H+‐transport were evaluated: by a mathematical model, describing the time‐course of H+‐gradient (ΔpH) formation; or by determining the rate of H+‐leakage following H+‐pumping inhibition by EDTA at the steady‐state ΔpH. When the H+‐transport rates were divided by those of PPi or ATP hydrolysis, measured under identical conditions, apparent stoichiometries of ca 2 were determined for the mitochondrial H+‐PPiase and H+‐ATPase, and for the vacuolar H+‐ATPase. The stoichiometry of the vacuolar H+‐PPiase was found to be ca 1. From these results, it is suggested that the mitochondrial H+‐PPiase may, in theory, function as a primary H+‐pump poised towards synthesis of PPi and, therefore, acting in parallel with the main H+‐ATPase.  相似文献   

4.
The effect of ADP on the activity of the plasma membrane (PM) H+‐ATPase of red beet ( Beta vulgaris L.) parenchyma discs was evaluated by analyzing the effect of increasing concentrations of ADP on the kinetics of the reaction. When the PM H+‐ATPase activity was assayed at pH 6.3, ADP behaved as a simple competitive inhibitor. When the activity was assayed at pH 7.1, ADP not only increased the apparent Km for MgATP but also decreased the Vmax of the reaction. When the C‐terminal domain of the PM H+‐ATPase was cleaved by controlled trypsin treatment or displaced by addition of lysophosphatidylcholine, only the competitive component of inhibition by ADP of the activity assayed at pH 7.1 was evident. The results are discussed in relation to the physiological relevance of the activation of the PM H+‐ATPase by displacement of the autoinhibitory C‐terminal domain.  相似文献   

5.
Erythrosin b, a potent inhibitor of the Ca2+‐ATPases and the Ca2+‐release channel (BCC1) in mechanosensitive tissue of Bryonia dioica Jacq., effectively suppresses a tendril's reaction to touch, suggesting that Ca2+‐transporters are involved in signal transduction in this organ. The Ca2+‐ATPase located in the endoplasmic reticulum (ER) represents a multiregulated enzyme that is stimulated by calmodulin (CaM), KCl and lysophospholipids. Limited proteolysis of ER‐membranes by trypsin results in an irreversible activation of the Ca2+‐ATPase and loss of the CaM sensitivity, presumably through removal of an autoinhibitory domain where CaM binds. Mild trypsination mimics the effects of CaM on Vmax and the affinity for Ca2+ and ATP. Irrespective of a trypsin treatment, the enzyme can be additionally stimulated by KCl and lysolipids, indicating that the sites of interaction for these effectors are not located in the domain removed by the protease. CaM‐stimulated ATPase activity was purified from microsomal and ER fractions using a combination of CaM‐affinity and anion‐exchange chromatography. The isolated polypeptide was enzymatically active, showed a calcium‐dependent mobility‐shift in SDS‐PAGE from 109 kDa in the absence of Ca2+ to 104 kDa in the presence of 10 m M CaCl2 and could be radiolabeled with [35S]‐CaM. The characteristics of the purified enzyme remained closely similar to those of the ER‐bound Ca2+‐transporting activity, including the enzymatic data, CaM stimulation, and the sensitivity towards a range of inhibitors.  相似文献   

6.
The cell and subcellular localization of plasma membrane P‐type H+‐ATPase in root apices from Zea mays L. (maize) seedlings was investigated by immunofluorescence microscopy. H+‐ATPase was highly abundant in cells of epidermal and endodermal tissues as well as in phloem companion cells. Strong immunodecoration was also observed in a subset of xylem parenchyma cells forming a connection between the endodermis and metaxylem. Evidence that these cells are equipped for active membrane transport raises the potential that they play a special role in xylem loading. Significant amounts of H+‐ATPase were also observed in outer cortical cells. Progressively less H+‐ATPase was seen in cortical cells further away from the root‐soil interface. The H+‐ATPase was asymmetrically localized within both epidermal and outer cortical cells, with higher levels detected on cell surfaces closest to the root‐soil interface. This asymmetric localization of H+‐ATPase is consistent with the hypothesis that transport systems for uptake of nutrients from the soil are selectively targeted to cell surfaces most exposed to nutrients.  相似文献   

7.
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   

8.
Abstract: We have shown previously that noradrenaline (NA) stimulated or inhibited the release of corticotropin-releasing hormone (CRH) according to the availability of adrenal steroids. The aim of the present work was to examine whether the changes in the NA modulation of CRH release from hypothalamic neurons result from a steroid-induced plasticity of the adrenergic transduction pathways. From anterior hypothalamic slices cultured in standard medium (i.e., containing adrenal steroids at a final dilution of 61 ± 9 ng/ml), (a) the stimulatory effect of NA on CRH release was reversed in a dose-dependent manner by increasing concentrations of the α1-adrenoreceptor antagonist prazosin, (b) activation of protein kinase C by acute treatment with phorbol 12-myristate 13-acetate (0.5 µ M , 1 h) mimicked NA stimulation of CRH secretion, and (c) the activation of L-type Ca2+ channels by Bay K 8644 also produce an increased CRH secretion. In contrast, the inhibitory effect of NA on CRH secretion from slices cultured in steroid-free medium was markedly reversed by the α2-adrenoreceptor antagonist yohimbine, by pretreatment with pertussin toxin, or by the addition of 4-aminopyridine, a K+-channel blocker. Acute treatment with phorbol 12-myristate 13-acetate did not change the inhibitory NA effect. Moreover, all these effects were reversed by daily corticosterone supplementation, for as long as they were tested. These results are consistent with a steroid-dependent change in the nature of adrenergic receptors and its associated transduction pathways involved in the regulation of CRH secretion in the hypothalamus.  相似文献   

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11.
Abstract: Ethanol dependence and tolerance involve perturbation of GABAergic neurotransmission. Previous studies have demonstrated that ethanol treatment regulates the function and expression of GABAA receptors throughout the CNS. Conceivably, changes in receptor function may be associated with alterations of subunit composition. In the present study, a comprehensive (1–12 weeks) ethanol treatment paradigm was used to evaluate changes in GABAA receptor subunit expression in several brain regions including the cerebellum, cerebral cortex, ventral tegmental area (VTA) (a region implicated in drug reward/dependence), and the hippocampus (a region involved in memory/cognition). Expression of α1 and α5 subunits was regulated by ethanol in a region-specific and time-dependent manner. Following 2–4 weeks of administration, cortical and cerebellar α1 and α5 subunit immunoreactivity was reduced. In the VTA, levels of α1 subunit immunoreactivity were significantly decreased after 12 weeks but not 1–4 weeks of treatment. Hippocampal α1 subunit immunoreactivity and mRNA content were also significantly reduced after 12 but not after 4 weeks of treatment. In contrast, α5 mRNA content was increased in this brain region. These data indicate that chronic ethanol administration alters GABAA receptor subunit expression in the VTA and hippocampus, effects that may play a role in the abuse potential and detrimental cognitive effects of alcohol.  相似文献   

12.
Abstract: The adrenergic regulation of histamine release was studied in rat brain slices labeled with L-[3H]histidine. Noradrenaline in increasing concentrations progressively inhibited K+-evoked [3H]histamine release from cortical slices, whereas phenylephrine and isoprenaline were ineffective. Yohimbine, a preferential α2-adrenoceptor antagonist, reversed the noradrenaline effect in an apparently competitive manner and with a mean K i value of 30 n M . Phentolamine reversed the noradrenaline effect with a similar potency, whereas propranolol was ineffective. The imidazolines clo-nidine and oxymetazoline acted as partial agonists, oxymeta-zoline even behaving as an apparent antagonist. In vivo clo-nidine also inhibited [3H]histamine formation in cerebral cortex, an effect reversed by the administration of yohimbine. However, yohimbine failed to increase significantly [3H]histamine release in vitro and [3H]histamine formation in vivo, suggesting that adrenergic receptors are not activated by endogenous noradrenaline released under basal conditions. It is concluded that adrenergic α2-adrenoceptors presumably located on histaminergic axons control release and synthesis of histamine in the brain.  相似文献   

13.
Abstract: In contrast to some other ion channels, there are at present no proteins known that bind specifically to mature GABAA receptor channels. Such proteins may be important for the structural organization and cytoskeletal anchoring of GABAA receptors and could also be expected to have channel modulatory effects. To identify proteins that are associated with naturally occurring GABAA receptors we immunoprecipitated these receptors from detergent-solubilized bovine brain membranes by an antibody directed against the α1-subunit. Tubulin and actin were observed to coprecipitate specifically with the receptors. Nine additional proteins were detected, hinting at a complex protein network associated with α1-subunit-containing GABAA receptors. Results of a biochemical characterization of these G ABAA receptor- t ubulin complex- a ssociated p roteins (GTAPs) are presented here. Peptide mass fingerprinting analysis and microsequencing of tryptic peptides indicated that at least three GTAPs have not been described until the present.  相似文献   

14.
Abstract: Chronic administration of ethanol to rats on an intermittent regimen, for 60 repeated intoxicating doses and repeated withdrawal episodes, results in a long-lasting kindling phenomenon. This involves an increasing severity of withdrawal, including a reduced threshold to seizures produced by the GABAA antagonist, pentylenetetrazol. We have shown previously that muscimol-evoked 36Cl efflux and paired-pulse inhibition (involving GABAA-mediated recurrent inhibition) were decreased persistently in the CA1 region of hippocampal slices from chronic intermittent ethanol (CIE)-treated rats. We now report elevated levels of mRNA in forebrain for the α4 subunit of the GABAA receptor (GABAR), considered to be a constituent of pharmacologically and physiologically novel subtypes of GABARs. Using in situ hybridization with digoxigenin-labeled RNA probes, we show that at 2 days withdrawal, 60-dose CIE leads to a significant 30% increase in α4 subunit mRNA levels in the dentate gyrus, 46% increase in the CA3, and 26% increase in the CA1 regions. In contrast, there was no significant change in the mRNAs for the α5 subunit or glutamic acid decarboxylase 67 in the same regions. This study suggests that GABAR subunit-selective alterations occur after CIE treatment, possibly resulting in the alteration of the subunit composition of GABARs, with presumably altered physiological functions. This plasticity of GABARs may contribute to the increased withdrawal severity, reduced hippocampal inhibition, and increased seizure susceptibility of this animal model of human alcohol dependence.  相似文献   

15.
Abstract: Epinephrine (Epi) mediates various physiological effects via α2A-adrenergic receptors (α2A-ARs). Studies in mice with a point mutation in the gene for α2A-AR have shown that these receptors are responsible for the centrally mediated depressor effects of α2-AR agonists. These studies underscore the importance of understanding the basic cellular mechanisms involved in the expression of α2A-ARs, of which little is known. We use astroglia cultured from the hypothalamus and brainstem of adult Sprague-Dawley rats as a model system in which to study factors that regulate α2A-AR expression. These cells contain α2-ARs, which are predominately of the α2A-AR subtype. Our studies have shown that Epi causes a dose- and time-dependent decrease in steady-state levels of α2A-AR mRNA and number of α2A-ARs, effects that are mediated via α1- and β-adrenergic receptors (α1-ARs and β-ARs). These effects of Epi on α2A-AR mRNA and α2A-AR number are mimicked by activation of protein kinase C or increases in cellular cyclic AMP, which are intracellular messengers activated by α1-ARs and β-ARs, respectively. Taken together, these results indicate that expression of α2A-ARs is regulated in a heterologous manner by Epi, via α1-AR- and β-AR-mediated intracellular pathways.  相似文献   

16.
Abstract: We found that the binding of [3H]prazosin, a selective ligand for α1-adrenergic recognition sites, is significantly lower in the frontal cortex of the genetically epilepsy-prone rats (GEPRs), as compared with normal Sprague-Dawley rats. Scatchard analysis reveals a decrease in the B max of [3H]prazosin binding with no change in the apparent K D, suggesting that there are fewer α1-adrenergic recognition sites in the frontal cortex of the GEPR. This abnormality is associated with a reduced capacity of norepinephrine (NE) to stimulate [3H]inositol monophosphate ([3H]IP1) formation in frontal cortex slices prelabeled with [3H]inositol. No significant differences in [3H]prazosin binding as well as NE-stimulated [3H]IP1 formation have been observed in other brain regions including hippocampus, corpus striatum, and inferior colliculus. These results indicate that a deficit in the α1-adrenergic receptor system in the frontal cortex may play a role in the seizure process in the GEPR.  相似文献   

17.
Abstract: 125I-α-Bungarotoxin (α-BGT) was used to characterize the binding sites for cholinergic ligands in lobster walking leg nerve membranes. The toxin binding component has been visualized histochemically on the external surfaces of intact axons and isolated axonal membrane fragments. Binding of α-BGT to nerve membrane preparations was demonstrated to be saturable and highly reversible ( K Dapp± 1.7 ± 0.32 × 10-7 M; B max± 249 ± 46 pmol/mg protein) at pH 7.8, 10 mM-Tris buffer. Binding showed a marked sensitivity to ionic strength that was attributable to the competitive effects of inorganic cations (particularly Ca2+ and Mg2+) in the medium. 125I-α-BGT binding could be inhibited by cholinergic drugs (atropine ≅ d -tubocurarine > nicotine > carbamylcholine ≅ choline) and local anesthetics (procaine > tetracaine = lidocaine), but was unaffected by other neuroactive compounds tested (e.g., tetrodotoxin, 4-aminopyridine, quinuclidinyl benzilate, octopamine, bicuculline, haloperidol, ouabain). The pharmacological sensitivity of toxin binding resembles that of nicotine binding to axonal membranes, but differs significantly from nicotinic cholinergic receptors described in neuromuscular junctions, fish electric organs, sympathetic ganglia, and the CNS. The possible physiological relevance of the axonal cholinergic binding component and its relationship to α-BGT binding sites in other tissues are discussed.  相似文献   

18.
The aim of this study was to identify the molecular genetic origin underlying the I variant of αs1-casein and to develop a DNA-based test for this polymorphism as a tool for genetic analyses independent of milk sample testing. All coding exons and flanking regions of the α s 1 -casein gene were sequenced in DNA samples from cattle of known α s 1 -casein genotypes ( BI , CI , II , CC ), determined by isoelectric focusing of milk samples. A nucleotide substitution (A>T) in exon 11 (g.19836A>T) leads to the exchange of Glu with Asp at amino acid position 84 of the mature protein (p.Glu84Asp) and perfectly co-segregated with the presence of the αs1-casein I variant in the milk of the analysed animals. Genotyping of a total of 680 DNA samples from 31 Bos taurus and Bos indicus cattle breeds and from Bos grunniens , Bison bison and Bison bonasus by restriction fragment length polymorphism analysis revealed the occurrence of Asp at position 84 at low frequencies in Bos taurus and Bos indicus breeds and established its origin from the α s1-casein C variant (p.Glu192Gly). Ten different intragenic haplotypes in the gene region from intron 8 to intron 12 were observed by sequencing, of which two occurred in Bison bison and one in Bison bonasus only. Using available casein gene complex information, an association of Asp at position 84 to β-casein A 2 and κ-casein B was shown in the Bos indicus breed Banyo Gudali . Taken together, we can postulate that the α s1-casein variant I is caused by a non-synonymous nucleotide substitution in exon 11 of the gene and that it originated within Bos indicus and spread to Bos taurus subsequently.  相似文献   

19.
Abstract: We have isolated from an American lobster ( Homarus americanus ) olfactory organ cDNA library a clone, hGαq, with >80% identity to mammalian and arthropod Gαq sequences. In brain and olfactory organ, hGαq mRNA was expressed predominantly in neurons, including virtually all the neuronal cell body clusters of the brain. Gαq protein was also expressed broadly, appearing on western blots as a single band of 46 kDa in brain, eyestalk, pereiopod, dactyl, tail muscle, olfactory organ, and aesthetasc hairs. These results suggest that hGαq plays a role in a wide variety of signal transduction events. Its presence in the olfactory aesthetasc hairs, which are almost pure preparations of the outer dendrites of the olfactory receptor neurons, the expression of a single hGαq mRNA species (6 kb) in the olfactory organ, and the localization of hGαq mRNA predominantly in the olfactory receptor neurons of the olfactory organ strongly suggest that one function of hGαq is to mediate olfactory transduction.  相似文献   

20.
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