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1.
The G-protein-gated inwardly rectifying K
+(GIRK) family of ion channels form functional Gβγ-sensitive channels as heteromultimers of GIRK1 and either the GIRK2 or GIRK4
subunits. However, the homologous mouse brain GIRK3 clone failed to express in the earliest reported functional experiments
in Xenopus oocytes. We recloned the GIRK3 subunit from mouse brain and found that the new clone differed significantly from that originally
reported. The functional aspects of GIRK3 were reinvestigated by expression in CHO cells. The single channel properties of
GIRK1/GIRK3 were characterized and compared to those of the GIRK1/GIRK2 and GIRK1/GIRK4 channels. All three GIRK1/GIRKx combinations
produced channels with nearly indistinguishable conductances and kinetics. The response of GIRK1/GIRK3 to Gβγ in the 1–47
nm range was examined and found to be indistinguishable from that of GIRK1/GIRK4 channels. We conclude that GIRK1, with either
GIRK2, 3, or 4, gives rise to heteromultimeric channels with virtually identical conductances, kinetics, and Gβγ sensitivities.
Received: 13 January 1999/Revised: 2 March 1999 相似文献
2.
β-Thalassemia is the most common single gene disorder in Iran and more than 25,000 affected individuals have been reported.
It has been reported that in patients with β-thalassemia in the presence of Xmn1 polymorphic site the level of Hb F and Gγ: Aγ ratio is increased. The prevalence of Xmn1 polymorphic site, Gγ: Aγ ratio and Hb F in 197 β-thalassemia major patients from the Kermanshah Province of Iran were studied. The Xmn1 polymorphic site was determined by PCR-RFLP procedure. The levels of Gγ and Aγ chains were detected by HPLC. The percent of Hb F was determined using electrophoresis method. In β-thalassemia major patients
the frequency of presence Xmn1 was 0.39. The mean of Gγ: Aγ ratio was found to be 2.5. In the present study it was found that in the presence of Xmn1 polymorphic site Gγ percent and Gγ: Aγ ratio were significantly increased (P = 0.01) and the clinical features such as splenomegaly and bone marrow expansion were significantly improved (P = 0.01). We found that in the presence of Xmn1 polymorphic site on both chromosomes (+/+) the level of Hb F tended to be increased compared to the absence of Xmn1 (−/−). The present investigation has studied the frequency of Xmn1 polymorphic site in β-thalassemia major patients from Western Iran and has revealed that the presence of this polymorphic
site caused a positive influence on Hb F production and the Gγ percent which could improve the clinical symptoms of β-thalassemia patients. 相似文献
3.
Methyl-detected ‘out-and-back’ NMR experiments for simultaneous assignments of Alaβ and Ileγ2 methyl groups in large proteins 总被引:1,自引:1,他引:0
A set of sensitive methyl-detected ‘out-and-back’ NMR experiments for simultaneous assignments of Alaβ and Ileγ2 methyl positions
in large proteins is described. The developed methodology is applied to an 82-kDa enzyme Malate Synthase G. Complete alanine
β and isoleucine γ2 1H–13C methyl chemical shift assignments could be obtained from the set of new methyl-detected ‘out-and-back’ 3D experiments. The
described methodology for methyl assignments should be applicable to protein molecules within approximately 100-kDa molecular
weight range irrespective of the labeling strategy chosen to produce selectively protonated Alaβ and Ileγ2 13CH3 sites on a deuterated background. 相似文献
4.
5.
Alkaline stable (pH 7.75–12.5) urease from Sporosarcina ureae was purified over 400-fold by ion exchange and hydrophobic interaction chromatography. The cytoplasmic enzyme was remarkably
active with a specific activity of greater than 9300 μmol urea degraded min-1 mg protein-1 at pH 7.5, where it has optimal activity. Although S. ureae is closely related to Bacillus pasteurii, known to posses a homopolymeric urease containing 1 nickel per subunit [M
r=65000], the S. ureae enzyme is comprised of three subunits [apparent M
r=63100 (α), 14500 (β), and 8500 (γ)] in an estimated ∝βγ stoichiometry and contains 2.1±0.6 nickel ions per ∝βγ unit as measured
by atomic absorption spectrometry. Stationary phase cultures sometimes possessed low levels of urease activity, but the specific
activity of cell extracts of partially purified urease preparations from such cultures could be elevated by heat treatment,
dilution, or dialysis to values comparable to those observed in samples from exponentially grown cells. 相似文献
6.
Currie KP 《Cellular and molecular neurobiology》2010,30(8):1201-1208
Catecholamines and other transmitters released from adrenal chromaffin cells play central roles in the “fight-or-flight” response
and exert profound effects on cardiovascular, endocrine, immune, and nervous system function. As such, precise regulation
of chromaffin cell exocytosis is key to maintaining normal physiological function and appropriate responsiveness to acute
stress. Chromaffin cells express a number of different G protein coupled receptors (GPCRs) that sense the local environment
and orchestrate this precise control of transmitter release. The primary trigger for catecholamine release is Ca2+ entry through voltage-gated Ca2+ channels, so it makes sense that these channels are subject to complex regulation by GPCRs. In particular G protein βγ heterodimers
(Gβγ) bind to and inhibit Ca2+ channels. Here I review the mechanisms by which GPCRs inhibit Ca2+ channels in chromaffin cells and how this might be altered by cellular context. This is related to the potent autocrine inhibition
of Ca2+ entry and transmitter release seen in chromaffin cells. Recent data that implicate an additional inhibitory target of Gβγ
on the exocytotic machinery and how this might fine tune neuroendocrine secretion are also discussed. 相似文献
7.
Miroslav Malešević Zsuzsanna Majer Elemér Vass Thomas Huber Ulf Strijowski Miklós Hollósi Norbert Sewald 《International journal of peptide research and therapeutics》2006,12(2):165-177
β-Amino acids with side chains at C2 and/or at C3 are of growing interest in drug design, as they may induce astonishing and unusual peptide conformations. Therefore it is of eminent importance to gather information on the consequences of β-amino acid incorporation on the three-dimensional structure of a peptide. This paper describes the synthesis and conformational analysis of cyclic penta- and hexapeptides comprising either (S)-Pro or (S)-β-Hpro. The conformational influence of the β-homoproline building block was analyzed by the combined application of CD, FT-IR and NMR. While the CD spectra of the proline containing peptides indicate the presence of inverse γ-turns and βII-turns, the CD spectra of the β-homoamino acid analogs are dominated by an unprecedented negative band near 205 nm associated with a pseudo-β-turn (Ψβ) or pseudo-γ-turn (Ψγ). These results were confirmed by FT-IR spectroscopy, which also indicates the formation of two internal hydrogen bonds in the cyclic peptides containing the β-homoproline. The conformations of the β-homoproline containing pentapeptides were additionally determined by NMR in combination with MD simulations in two different solvents. The conformation in trifluoroethanol (TFE) is characterized by a bifurcated hydrogen bond stabilizing a pseudo-γ-turn with β-homoproline in the central position, nested with a pseudo-β-turn with β-homoproline in the i+1 position. The combined CD/FT-IR studies clearly show that the replacement of proline by β-homoproline gives rise to a more flexible peptide backbone, and CD spectroscopy hints towards the presence of pseudo-β- or pseudo-γ-turns. 相似文献
8.
9.
Methyl 13CHD2 isotopomers of all methyl-containing amino-acids can be observed in residually protonated samples of large proteins obtained
from [U-13C,1H]-glucose/D2O-based bacterial media, with sensitivity sufficient for a number of NMR applications. Selective detection of some subsets
of methyl groups (Alaβ, Thrγ2) is possible using simple ‘out-and-back’ NMR methodology. Such selective methyl-detected ‘out-and-back’ NMR experiments allow
complete assignments of threonine γ2 methyls in residually protonated, [U-13C,1H]-glucose/D2O-derived samples of an 82-kDa enzyme Malate Synthase G. [U-13C,1H]-glucose/D2O-derived protein samples are relatively inexpensive and are usually available at very early stages of any NMR study of high-molecular-weight
systems. 相似文献
10.
W. E. Rodriguez Romero M. Castillo M. A. Chaves G. F. Saenz L.-H. Gu J. B. Wilson E. Baysal N. S. Smetanina J. Y. Leonova T. H. J. Huisman 《Human genetics》1996,97(6):829-833
We have identified a minor hemoglobin component (∼5%) in the blood of a healthy Costa Rican female, but not in her mother
and two brothers (father not studied), that has an His→Arg replacement at position β77 (Hb Costa Rica). No other amino acid
replacements were observed and no β- or γ-chain-like peptides were present. Hb Costa Rica has a normal stability. Sequence
analyses of numerous polymerase chain reaction (PCR)-amplified segments of DNA that contain exon 2 of the β gene failed to
identify a CAC→CGC (His→Arg) mutation. The same was the case when cDNA was sequenced, indicating that a β-Costa Rica-mRNA could not be detected
with this procedure. Gene mapping of genomic DNA with BglII, BamHI, and HindIII gave normal fragments only and with the same intensity as observed for the fragments of a normal control. The quantities
of the β chain variants Hb J-Iran and Hb Fukuyama with related mutations at β77 vary between 30% and 45% in heterozygotes,
whereas that of Hb F-Kennestone with the same His→Arg mutation but in the Gγ-globin gene, is a high 40%–45% (as percentage of total Gγ) in a heterozygous newborn. These different observations exclude a heterozygosity of the A→G mutation at codon β77, as well
as a deletion comparable to that of Hbs Lepore or Kenya, or a β-globin gene duplication, and point to a nontraditional inheritance
of Hb Costa Rica. Allele-specific amplification of cDNA with appropriate primers identified the presence of a low level of
mutated mRNA in the reticulocytes of the patient, which was confirmed by dotblot analysis of the same material with 32P-labeled probes. Comparable amplification products were not observed in genomic DNA. The A→G mutation apparently occurred
in a somatic cell at a relatively early stage in the development of the hematopoietic cell system, and Hb Costa Rica accumulated
through rapid cell divisions in patchy areas in the bone marrow (somatic mosaicism). An unequal distribution of Hb Costa Rica
over the red cells supports this possibility.
Received: 25 August 1995 / Revised: 13 December 1995 相似文献
11.
The sequence specific 1H, 13C, and 15N resonance assignments of Hahellin, a putative member of βγ-crystallin family, from Hahella Chejuensis, have been accomplished by NMR spectroscopy. The resonance assignments reveal that the protein adopts predominantly a β-sheet
conformation as in the case of βγ-crystallin folds. 相似文献
12.
Shinya Nagafuchi Mamoru Totsuka Satoshi Hachimura Masao Goto Takeshi Takahashi Takaji Yajima Tamotsu Kuwata Shuichi Kaminogawa 《Cytotechnology》2002,40(1-3):49-58
We have investigated the influence of dietary nucleotides on the intestinal immune system in ovalbumin (OVA)-specific T-cell
receptor (TCR) transgenic mice (OVA-TCR Tg mice). When mice were supplied with water supplemented with 2% OVA ad libitum, the faecal OVA-specific immunoglobulin A (IgA) level significantly increased in those fed a nucleotide-supplemented diet
(NT(+) diet) compared with those fed a nucleotide-free control diet (NT(–) diet). In the NT(+) diet-fed mice, secretion of
transforming growth factor β (TGF-β), which is an isotype-specific switch factor for IgA, from intestinal epithelial cells
(IECs) was significantly increased. Furthermore, an increased proportion of intestinal intraepithelial lymphocytes (IELs)
bearing γδ TCR (TCRγδ+ IELs) and increased secretion from IECs of interleukin 7 (IL-7), which is essential for the development of TCRγδ+ IELs, were also observed in OVA-TCR-Tg mice fed the NT(+) diet, as we previously demonstrated using BALB/c mice (Nagafuchi
et al., Biosci. Biotechnol. Biochem. 64: 1459-65 (2000)). Considering that TCRγδ+ T cells and TGF-β are important for an induction of the mucosal IgA response, our results suggest that dietary nucleotides
augment the mucosal OVA-specific IgA response by increasing the secretion of TGF-β from IECs and the proportion of TCRγδ+ IELs.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
13.
Sandoval A Arikkath J Monjaraz E Campbell KP Felix R 《Cellular and molecular neurobiology》2007,27(7):901-908
(1) Voltage-gated Ca2+ (CaV) channels are multi-subunit membrane complexes that allow depolarization-induced Ca2+ influx into cells. The skeletal muscle L-type CaV channels consist of an ion-conducting CaV1.1 subunit and auxiliary α2δ−1, β1 and γ1 subunits. This complex serves both as a CaV channel and as a voltage sensor for excitation–contraction coupling. (2) Though much is known about the mechanisms by which
the α2δ−1 and β1 subunits regulate CaV channel function, there is far less information on the γ1 subunit. Previously, we characterized the interaction of γ1 with the other components of the skeletal CaV channel complex, and showed that heterologous expression of this auxiliary subunit decreases Ca2+ current density in myotubes from γ1 null mice. (3) In the current report, using Western blotting we show that the expression of the CaV1.1 protein is significantly lower when it is heterologously co-expressed with γ1. Consistent with this, patch-clamp recordings showed that transient transfection of γ1 drastically inhibited macroscopic currents through recombinant N-type (CaV2.2/α2δ−1/β3) channels expressed in HEK-293 cells. (4) These findings provide evidence that co-expression of the auxiliary γ1 subunit results in a decreased expression of the ion-conducting subunit, which may help to explain the reduction in Ca2+ current density following γ1 transfection. 相似文献
14.
We previously described a novel mode of downregulation of human immunodeficiency virus type 1 (HIV-1) Gag expression by a
cytoplasmic domain fusion protein of the envelope (Env) transmembrane protein, β-galactosidase (β-gal)/706–856, which contains
the cytoplasmic tail of gp41 fused at the C terminus of Escherichia coli β-gal. In the present study, we showed that this mediator conferred a dose-dependent dominant interference with virus infectivity.
In the context of an HIV-1 provirus, this inhibitor downregulated steady-state Env expression. Paradoxically, Env overexpression
suppressed β-gal/706–856-mediatd Gag downregulation. Sucrose gradient ultracentrifugation and confocal microscopy revealed
that Gag, Env, and β-gal/706–856 had stable interactions and formed aggregated complexes in perinuclear regions. Moreover,
Env overexpression hindered colocalization of Gag with β-gal/706–856 in the perinuclear region. Further cytoplasmic domain
mapping analyses showed a correlation between the ability of cytoplasmic subdomains to downregulate Gag expression and the
ability of these subdomains to stably interact with Gag. These studies show that redirection of Gag from its cytoplasmic synthesis
site to a perinuclear compartment is a prerequisite for β-gal/706–856-mediated Gag downregulation. The results also illustrate
that the dynamic interplay among Gag, Env, and β-gal/706–856 can modulate Gag and Env expression, thus controlling HIV-1 infection. 相似文献
15.
Ningzhi Xu Omar Coso Daruka Mahadevan Antonio De Blasi Paul K. Goldsmith William F. Simonds J. Silvio Gutkind 《Cellular and molecular neurobiology》1996,16(1):51-59
Summary 1. The noncatalytic domain of Ras-GAP can affect signaling through G protein-coupled receptors by a poorly understood mechanism.
2. In this study, fusion proteins containing elements of the noncatalytic domain ofras-GAP were examined for their ability to bindβγ subunits of heterotrimeric G proteins and phosphotyrosine-containing polypeptides.
3. Our results demonstrate that purifiedβγ dimers associated with bacterially expressed GAP proteins and that this association does not require SH2 or SH3 domains but
is dependent on the presence of the GAP pleckstrin-homology (PH) domain. In contrast, only the SH2 domains are necessary for
binding to tyrosine phosphorylated proteins.
4. These findings raise the possibility that heterotrimeric G proteins might affect functioning ofras-like proteins throughβγ subunits acting on their regulatory molecules. 相似文献
16.
H. J. Carlisle T. G. Hales B. A. Schlinger 《Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology》1998,182(4):531-538
Songbirds are widely studied to investigate the hormonal control of behavior. However, little is known about the effects
of steroids on neurotransmission in these birds. We used electrophysiological and pharmacological techniques to characterize
γ-aminobutyric acid (GABA) type A receptors (GABAA) of primary cultured telencephalic and hippocampal neurons from developing zebra finches. Additionally, their modulation
by 17β-estradiol(E2), 5α- and 5β-dihydrotestosterone (DHT), 5α- and 5β-pregnan-3α-ol-20-one, and corticosterone was examined. Whole-cell GABA-evoked
currents were inhibited by picrotoxin (10 μmol l−1) and bicuculline methiodide (10 μmol l−1) and potentiated by pentobarbital (100 μmol l−1) and propofol (3 μmol l−1). Loreclezole (10 μmol l−1) potentiated GABA-evoked currents, suggesting the presence of β2, β3 and/or β4 subunits. Diazepam (1 μmol l−1) potentiated currents, while Zn2+ (1 μmol l−1) caused no inhibition, indicating the presence of γ subunits. 5α- and 5β-Pregnan-3α-ol-20-one (100 nmol l−1) potentiated currents, whereas E2 (1 μmol l−1), 5α- and 5β-DHT (1 μmol l−1), and corticosterone (10 μmol l−1) had no detectable effect. We conclude that zebra finch telencephalic and hippocampal GABAA receptors include α, β, and γ subunits and are similar to their mammalian counterparts in both their biophysical and pharmacological
properties. Additionally, GABA-evoked currents are greatly potentiated by 5α- and 5β-pregnan-3α-ol-20-one but show little
or no acute modulation by sex steroids or corticosterone.
Accepted: 12 November 1997 相似文献
17.
Alesutan I Sopjani M Munoz C Fraser S Kemp BE Föller M Lang F 《The Journal of membrane biology》2011,240(3):151-158
Connexins provide intercellular connections that allow passage of ions and small organic molecules. They clamp the cell membrane
potential and cellular ion composition to that of neighboring cells. The cell membrane potential and ion composition of an
energy-depleted cell could thus be maintained despite its compromised Na+/K+ activity. By the same token, however, the breakdown of ion gradients in that cell imposes an additional challenge to the
neighboring cells, which may jeopardize their survival. Thus, timely closure of connexins may be critically important for
the survival of those cells. Energy depletion stimulates the AMP-activated protein kinase (AMPK), a serine/threonine kinase
that senses energy depletion and stimulates several cellular mechanisms to enhance energy production and to limit energy utilization.
The present study explored whether AMPK regulates connexin 26. To this end, cRNA encoding connexin 26 was injected into Xenopus oocytes with and without additional injection of wild-type AMPK (α1β1γ1), of the constitutively active γR70QAMPK (α1β1γ1[R70Q]) or of the inactive mutant αK45RAMPK (α1[K45R]β1γ1). Connexin 26 activity was determined in dual-electrode voltage-clamp experiments. Moreover, connexin 26
abundance was determined in the oocyte cell membrane by chemiluminescence and confocal microscopy. As a result, connexin 26-mediated
current and connexin 26 protein abundance were significantly decreased by coexpression of γR70QAMPK and, to a lower extent, by wild-type AMPK but not by αK45RAMPK. In conclusion, AMPK is a potent regulator of connexin 26. 相似文献
18.
An Unstructured Region is Required by GAV Homologue
for the Fibrillization of Host Proteins 总被引:3,自引:0,他引:3
Accumulating evidence shows that some amyloidogenic proteins contain core sequences, which are critical for their fibrillization.
Core sequences of α-synuclein, β-amyloid peptide and prion protein usually reside in their unfolded regions and share a conserved
consensus (VGGAVVAGV) designated as GAV homologue. Here we investigate the role of unfolded regions in fibrillization after
GAV homologue is attached to the C-terminus or inserted into the loop regions of different host proteins, namely α -Syn1-65, γ-synuclein, E. coli thioredoxin and immunoglobulin G binding B1 domain of streptococcal protein G. The results imply that an unstructured region is required by GAV homologue for the fibrillization of host proteins.
A number of amyloidogenic proteins with core sequences located in unstructured regions are summarized and discussed in details.
The finding may provide further insight into the elucidating of the molecular mechanism underlying the fibrillization of α-Syn,
Aβ and PrP as well as other amyloidogenic proteins. 相似文献
19.
Arnaud Bianchi David Moulin Sylvie Sebillaud Meriem Koufany Marie-Madeleine Galteau Patrick Netter Bernard Terlain Jean-Yves Jouzeau 《Arthritis research & therapy》2005,7(6):R1325
Microsomal prostaglandin E synthase (mPGES)-1 is a newly identified inducible enzyme of the arachidonic acid cascade with
a key function in prostaglandin (PG)E2 synthesis. We investigated the kinetics of inducible cyclo-oxygenase (COX)-2 and mPGES-1 expression with respect to the production
of 6-keto-PGF1α and PGE2 in rat chondrocytes stimulated with 10 ng/ml IL-1β, and compared their modulation by peroxisome-proliferator-activated receptor
(PPAR)γ agonists. Real-time PCR analysis showed that IL-1β induced COX-2 expression maximally (37-fold) at 12 hours and mPGES-1
expression maximally (68-fold) at 24 hours. Levels of 6-keto-PGF1α and PGE2 peaked 24 hours after stimulation with IL-1β; the induction of PGE2 was greater (11-fold versus 70-fold, respectively). The cyclopentenone 15-deoxy-Δ12,14prostaglandin J2 (15d-PGJ2) decreased prostaglandin synthesis in a dose-dependent manner (0.1 to 10 μM), with more potency on PGE2 level than on 6-keto-PGF1α level (-90% versus -66% at 10 μM). A high dose of 15d-PGJ2 partly decreased COX-2 expression but decreased mPGES-1 expression almost completely at both the mRNA and protein levels.
Rosiglitazone was poorly effective on these parameters even at 10 μM. Inhibitory effects of 10 μM 15d-PGJ2 were neither reduced by PPARγ blockade with GW-9662 nor enhanced by PPARγ overexpression, supporting a PPARγ-independent
mechanism. EMSA and TransAM? analyses demonstrated that mutated IκBα almost completely suppressed the stimulating effect of IL-1β on mPGES-1 expression
and PGE2 production, whereas 15d-PGJ2 inhibited NF-κB transactivation. These data demonstrate the following in IL-1-stimulated rat chondrocytes: first, mPGES-1
is rate limiting for PGE2 synthesis; second, activation of the prostaglandin cascade requires NF-κB activation; third, 15d-PGJ2 strongly inhibits the synthesis of prostaglandins, in contrast with rosiglitazone; fourth, inhibition by 15d-PGJ2 occurs independently of PPARγ through inhibition of the NF-κB pathway; fifth, mPGES-1 is the main target of 15d-PGJ2. 相似文献
20.
Aromatic proton resonances of proteins are notoriously difficult to assign. Through-bond correlation experiments are preferable
over experiments that rely on through-space interactions because they permit aromatic chemical shift assignments to be established
independently of the structure determination process. Known experimental schemes involving a magnetization transfer across
the Cβ–Cγ bond in aromatic side chains either suffer from low efficiency for the relay beyond the Cδ position, use sophisticated 13C mixing schemes, require probe heads suitable for application of high 13C radio-frequency fields or rely on specialized isotopic labelling patterns. Novel methods are proposed that result in sequential
assignment of all aromatic protons in uniformly 13C/15N labelled proteins using standard spectrometer hardware. Pulse sequences consist of routinely used building blocks and are
therefore reasonably simple to implement. Ring protons may be correlated with β-carbons and, alternatively, with amide protons
(and nitrogens) or carbonyls in order to take advantage of the superior dispersion of backbone resonances. It is possible
to record spectra in a non-selective manner, yielding signals of all aromatic residues, or as amino-acid type selective versions
to further reduce ambiguities. The new experiments are demonstrated with four different proteins with molecular weights ranging
from 11 kDa to 23 kDa. Their performance is compared with that of (Hβ)Cβ(CγCδ)Hδ and (Hβ)Cβ(CγCδCɛ)Hɛ pulse sequences [Yamazaki
et al. (1993) J Am Chem Soc 115:11054–11055].
Electronic Supplementary Material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. 相似文献