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Electron spin resonance (ESR) studies of radicals formed by radiation-induced multiple one-electron oxidations of guanine moieties in DNA are reported in this work. Annealing of gamma-irradiated DNA from 77 to 235 K results in the hydration of one electron oxidized guanine (G•+) to form the 8-hydroxy-7,8-dihydroguanin-7-yl-radical (•GOH) having one β-proton coupling of 17–28 G and an anisotropic nitrogen coupling, A‖, of ~20 G, A = 0 with g‖ = 2.0026 and g = 2.0037. Further annealing to 258 K results in the formation of a sharp singlet at g = 2.0048 with line-width of 5.3 G that is identified as the 8-oxo-7,8-dihydroguanine one-electron-oxidized radical (8-oxo-G•+). This species is formed via two one-electron oxidations of •GOH. These two one-electron oxidation steps leading to the formation of 8-oxo-G•+ from •GOH in DNA, are in accordance with the expected ease of oxidation of •GOH and 8-oxo-G. The incorporation of oxygen from water in G•+ leading to •GOH and to 8-oxo-G•+ is verified by ESR studies employing 17O isotopically enriched water, which provide unambiguous evidence for the formation of both radicals. ESR analysis of irradiated-DNA in the presence of the electron scavenger, Tl3+, demonstrates that the cationic pathway leads to the formation of the 8-oxo-G•+. In irradiated DNA–Tl3+ samples, Tl3+ captures electrons. Tl2+ thus produced is a strong oxidant (2.2 V), which is metastable at 77 K and is observed to increase the formation of G•+ and subsequently of 8-oxo-G•+ upon annealing. We find that in the absence of the electron scavenger the yield of 8-oxo-G•+ is substantially reduced as a result of electron recombinations with G•+ and possible reaction with •GOH. 相似文献
3.
Zhang Y Liu L Wu X An X Stubbe J Huang M 《The Journal of biological chemistry》2011,286(48):41499-41509
The β2 subunit of class Ia ribonucleotide reductase (RNR) contains a diferric tyrosyl radical cofactor (Fe2III-Tyr•) that is essential for nucleotide reduction. The β2 subunit of Saccharomyces cerevisiae is a heterodimer of Rnr2 (β) and Rnr4 (β′). Although only β is capable of iron binding and Tyr• formation, cells lacking β′ are either dead or exhibit extremely low Tyr• levels and RNR activity depending on genetic backgrounds. Here, we present evidence supporting the model that β′ is required for iron loading and Tyr• formation in β in vivo via a pathway that is likely dependent on the cytosolic monothiol glutaredoxins Grx3/Grx4 and the Fe-S cluster protein Dre2. rnr4 mutants are defective in iron loading into nascent β and are hypersensitive to iron depletion and the Tyr•-reducing agent hydroxyurea. Transient induction of β′ in a GalRNR4 strain leads to a concomitant increase in iron loading and Tyr• levels in β. Tyr• can also be rapidly generated using endogenous iron when permeabilized Δrnr4 spheroplasts are supplemented with recombinant β′ and is inhibited by adding an iron chelator prior to, but not after, β′ supplementation. The growth defects of rnr4 mutants are enhanced by deficiencies in grx3/grx4 and dre2. Moreover, depletion of Dre2 in GalDRE2 cells leads to a decrease in both Tyr• levels and ββ′ activity. This result, in combination with previous findings that a low level of Grx3/4 impairs RNR function, strongly suggests that Grx3/4 and Dre2 serve in the assembly of the deferric Tyr• cofactor in RNR. 相似文献
4.
Sugiura M Ogami S Kusumi M Un S Rappaport F Boussac A 《The Journal of biological chemistry》2012,287(16):13336-13347
The main cofactors that determine the photosystem II (PSII) oxygen evolution
activity are borne by the D1 and D2 subunits. In the cyanobacterium
Thermosynechococcus elongatus, there are three
psbA genes coding for D1. Among the 344 residues
constituting D1, there are 21 substitutions between PsbA1 and PsbA3, 31 between
PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. Here, we present the first
study of PsbA2-PSII. Using EPR and UV-visible time-resolved absorption
spectroscopy, we show that: (i) the time-resolved EPR spectrum of TyrZ• in the
(S3TyrZ•)′ is slightly modified; (ii) the split EPR signal
arising from TyrZ• in the (S2TyrZ•)′ state induced by near-infrared
illumination at 4.2 K of the S3TyrZ state is significantly
modified; and (iii) the slow phases of P680+⋅ reduction by TyrZ are
slowed down from the hundreds of μs time range to the ms time range,
whereas both the S1TyrZ• → S2TyrZ and
the S3TyrZ• → S0TyrZ + O2
transition kinetics remained similar to those in PsbA(1/3)-PSII. These results
show that the geometry of the TyrZ phenol and its environment, likely
the Tyr-O···H···Nϵ-His bonding,
are modified in PsbA2-PSII when compared with PsbA(1/3)-PSII. They also point to
the dynamics of the proton-coupled electron transfer processes associated with
the oxidation of TyrZ being affected. From sequence comparison, we
propose that the C144P and P173M substitutions in PsbA2-PSII
versus PsbA(1/3)-PSII, respectively located upstream of the
α-helix bearing TyrZ and between the two α-helices
bearing TyrZ and its hydrogen-bonded partner, His-190, are
responsible for these changes. 相似文献
5.
When skeletal muscles are activated and mechanically shortened, the force that is produced by the muscle fibers decreases in two phases, marked by two changes in slope (P1 and P2) that happen at specific lengths (L1 and L2). We tested the hypothesis that these force transients are determined by the amount of myosin cross-bridges attached to actin and by changes in cross-bridge strain due to a changing fraction of cross-bridges in the pre-power-stroke state. Three separate experiments were performed, using skinned muscle fibers that were isolated and subsequently (i) activated at different Ca2+ concentrations (pCa2+ 4.5, 5.0, 5.5, 6.0) (n = 13), (ii) activated in the presence of blebbistatin (n = 16), and (iii) activated in the presence of blebbistatin at varying velocities (n = 5). In all experiments, a ramp shortening was imposed (amplitude 10%Lo, velocity 1 Lo•sarcomere length (SL)•s−1), from an initial SL of 2.5 µm (except by the third group, in which velocities ranged from 0.125 to 2.0 Lo•s−1). The values of P1, P2, L1, and L2 did not change with Ca2+ concentrations. Blebbistatin decreased P1, and it did not alter P2, L1, and L2. We developed a mathematical cross-bridge model comprising a load-dependent power-stroke transition and a pre-power-stroke cross-bridge state. The P1 and P2 critical points as well as the critical lengths L1 and L2 were explained qualitatively by the model, and the effects of blebbistatin inhibition on P1 were also predicted. Furthermore, the results of the model suggest that the mechanism by which blebbistatin inhibits force is by interfering with the closing of the myosin upper binding cleft, biasing cross-bridges into a pre-power-stroke state. 相似文献
6.
Background
Within the animal kingdom, horses are among the most powerful aerobic athletic mammals. Determination of muscle respiratory capacity and control improves our knowledge of mitochondrial physiology in horses and high aerobic performance in general.Methodology/Principal Findings
We applied high-resolution respirometry and multiple substrate-uncoupler-inhibitor titration protocols to study mitochondrial physiology in small (1.0–2.5 mg) permeabilized muscle fibres sampled from triceps brachii of healthy horses.Oxidative phosphorylation (OXPHOS) capacity (pmol O2•s−1•mg−1 wet weight) with combined Complex I and II (CI+II) substrate supply (malate+glutamate+succinate) increased from 77±18 in overweight horses to 103±18, 122±15, and 129±12 in untrained, trained and competitive horses (N = 3, 8, 16, and 5, respectively). Similar to human muscle mitochondria, equine OXPHOS capacity was limited by the phosphorylation system to 0.85±0.10 (N = 32) of electron transfer capacity, independent of fitness level. In 15 trained horses, OXPHOS capacity increased from 119±12 to 134±37 when pyruvate was included in the CI+II substrate cocktail. Relative to this maximum OXPHOS capacity, Complex I (CI)-linked OXPHOS capacities were only 50% with glutamate+malate, 64% with pyruvate+malate, and 68% with pyruvate+malate+glutamate, and ∼78% with CII-linked succinate+rotenone. OXPHOS capacity with glutamate+malate increased with fitness relative to CI+II-supported ETS capacity from a flux control ratio of 0.38 to 0.40, 0.41 and 0.46 in overweight to competitive horses, whereas the CII/CI+II substrate control ratio remained constant at 0.70. Therefore, the apparent deficit of the CI- over CII-linked pathway capacity was reduced with physical fitness.Conclusions/Significance
The scope of mitochondrial density-dependent OXPHOS capacity and the density-independent (qualitative) increase of CI-linked respiratory capacity with increased fitness open up new perspectives of integrative and comparative mitochondrial respiratory physiology. 相似文献7.
Background
Mechlorethamine [ClCH2CH2N(CH3)CH2CH2Cl], a nitrogen mustard alkylating agent, has been proven to form a DNA interstrand crosslink at a cytosine-cytosine (C-C) mismatch pair using gel electrophoresis. However, the atomic connectivity of this unusual crosslink is unknown.Methodology/Principal Findings
HPLC-UV, MALDI-TOF-MS, and ESI-MS/MS were used to determine the atomic connectivity of the DNA C-C crosslink formed by mechlorethamine, MALDI-TOF-MS of the HPLC-purified reaction product of mechlorethamine with the DNA duplex d[CTCACACCGTGGTTC]•d[GAACCACCGTGTGAG] (underlined bases are a C-C mismatch pair) indicated formation of an interstrand crosslink at m/z 9222.088 [M−2H+Na]+. Following enzymatic digestion of the crosslinked duplex by snake venom phosphodiesterase and calf intestinal phosphatase, ESI-MS/MS indicated the presence of dC-mech-dC [mech = CH2CH2N(CH3)CH2CH2] at m/z 269.2 [M]2+ (expected m/z 269.6, exact mass 539.27) and its hydrolytic product dC-mech-OH at m/z 329.6 [M]+ (expected m/z 329.2). Fragmentation of dC-mech-dC gave product ions at m/z 294.3 and 236.9 [M]+, which are both due to loss of the 4-amino group of cytosine (as ammonia), in addition to dC and dC+HN(CH3)CH = CH2, respectively. The presence of m/z 269.2 [M]2+ and loss of ammonia exclude crosslink formation at cytosine N4 or O2 and indicate crosslinking through cytosine N3 with formation of two quaternary ammonium ions.Conclusions
Our results provide an important addition to the literature, as the first example of the use of HPLC and MS for analysis of a DNA adduct at the N3 position of cytosine. 相似文献8.
9.
10.
Macrophage-derived radicals generated by the NADPH oxidase complex and inducible nitric-oxide synthase (iNOS) participate in cytotoxic mechanisms against microorganisms. Nitric oxide (•NO) plays a central role in the control of acute infection by Trypanosoma cruzi, the causative agent of Chagas disease, and we have proposed that much of its action relies on macrophage-derived peroxynitrite (ONOO− + ONOOH) formation, a strong oxidant arising from the reaction of •NO with superoxide radical (O2˙̄). Herein, we have shown that internalization of T. cruzi trypomastigotes by macrophages triggers the assembly of the NADPH oxidase complex to yield O2˙̄ during a 60–90-min period. This does not interfere with IFN-γ-dependent iNOS induction and a sustained •NO production (∼24 h). The major mechanism for infection control via reactive species formation occurred when •NO and O2˙̄ were produced simultaneously, generating intraphagosomal peroxynitrite levels compatible with microbial killing. Moreover, biochemical and ultrastructural analysis confirmed cellular oxidative damage and morphological disruption in internalized parasites. Overexpression of cytosolic tryparedoxin peroxidase in T. cruzi neutralized macrophage-derived peroxynitrite-dependent cytotoxicity to parasites and favored the infection in an animal model. Collectively, the data provide, for the first time, direct support for the action of peroxynitrite as an intraphagosomal cytotoxin against pathogens and the premise that microbial peroxiredoxins facilitate infectivity via decomposition of macrophage-derived peroxynitrite. 相似文献
11.
Tang TT Zhu ZF Wang J Zhang WC Tu X Xiao H Du XL Xia JH Dong NG Su W Xia N Yan XX Nie SF Liu J Zhou SF Yao R Xie JJ Jevallee H Wang X Liao MY Shi GP Fu M Liao YH Cheng X 《PloS one》2011,6(9):e24272
Objective
Animal studies suggest that regulatory T (Treg) cells play a beneficial role in ventricular remodeling and our previous data have demonstrated defects of Treg cells in patients with chronic heart failure (CHF). However, the mechanisms behind Treg-cell defects remained unknown. We here sought to elucidate the mechanism of Treg-cell defects in CHF patients.Methods and Results
We performed flow cytometry analysis and demonstrated reduced numbers of peripheral blood CD4+CD25+FOXP3+CD45RO−CD45RA+ naïve Treg (nTreg) cells and CD4+CD25+FOXP3+CD45RO+CD45RA− memory Treg (mTreg) cells in CHF patients as compared with non-CHF controls. Moreover, the nTreg/mTreg ratio (p<0.01), CD4+CD25+FOXP3+CD45RO− CD45RA+CD31+ recent thymic emigrant Treg cell (RTE-Treg) frequency (p<0.01), and T-cell receptor excision circle levels in Treg cells (p<0.01) were lower in CHF patients than in non-CHF controls. Combined annexin-V and 7-AAD staining showed that peripheral Treg cells from CHF patients exhibited increased spontaneous apoptosis and were more prone to interleukin (IL)-2 deprivation- and CD95 ligand-mediated apoptosis than those from non-CHF individuals. Furthermore, analyses by both flow cytometry and real-time polymerase chain reaction showed that Treg-cell frequency in the mediastinal lymph nodes or Foxp3 expression in hearts of CHF patients was no higher than that of the non-CHF controls.Conclusion
Our data suggested that the Treg-cell defects of CHF patients were likely caused by decreased thymic output of nascent Treg cells and increased susceptibility to apoptosis in the periphery. 相似文献12.
Background
In contrast to intestinal CD4+ regulatory T cells (Tregs), the generation and function of immunomodulatory intestinal CD8+ T cells is less well defined. To dissect the immunologic mechanisms of CD8+ T cell function in the mucosa, reactivity against hemagglutinin (HA) expressed in intestinal epithelial cells of mice bearing a MHC class-I-restricted T-cell-receptor specific for HA was studied.Methodology and Principal Findings
HA-specific CD8+ T cells were isolated from gut-associated tissues and phenotypically and functionally characterized for the expression of Foxp3+ and their suppressive capacity. We demonstrate that intestinal HA expression led to peripheral induction of HA-specific CD8+Foxp3+ T cells. Antigen-experienced CD8+ T cells in this transgenic mouse model suppressed the proliferation of CD8+ and CD4+ T cells in vitro. Gene expression analysis of suppressive HA-specific CD8+ T cells revealed a specific up-regulation of CD103, Nrp1, Tnfrsf9 and Pdcd1, molecules also expressed on CD4+ Treg subsets. Finally, gut-associated dendritic cells were able to induce HA-specific CD8+Foxp3+ T cells.Conclusion and Significance
We demonstrate that gut specific antigen presentation is sufficient to induce CD8+ Tregs in vivo which may maintain intestinal homeostasis by down-modulating effector functions of T cells. 相似文献13.
M-DNA is a complex between the divalent metal ions Zn2+, Ni2+ and Co2+ and duplex DNA which forms at a pH of ~8.5. The stability and formation of M-DNA was monitored with an ethidium fluorescence assay in order to assess the relationship between pH, metal ion concentration, DNA concentration and the base composition. The dismutation of calf thymus DNA exhibits hysteresis with the formation of M-DNA occurring at a higher pH than the reconversion of M-DNA back to B-DNA. Hysteresis is most prominent with the Ni form of M-DNA where complete reconversion to B-DNA takes several hours even in the presence of EDTA. Increasing the DNA concentration leads to an increase in the metal ion concentration required for M-DNA formation. Both poly(dG)•poly(dC) and poly(dA)•poly(dT) formed M-DNA more readily than the corresponding mixed sequence DNAs. For poly(dG)•(poly(dC) M-DNA formation was observed at pH 7.4 with 0.5 mM ZnCl2. Modified bases were incorporated into a 500 bp fragment of phage λ DNA by polymerase chain reaction. DNAs in which guanine was replaced with hypoxanthine or thymine with 5-fluorouracil formed M-DNA at pHs below 8 whereas substitutions such as 2-aminoadenine and 5-methylcytosine had little effect. Poly[d(A5FU)] also formed a very stable M-DNA duplex as judged from Tm measurements. It is evident that the lower the pKa of the imino proton of the base, the lower the pH at which M-DNA will form; a finding that is consistent with the replacement of the imino proton with the metal ion. 相似文献
14.
Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca2+ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca2+ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP3Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP3Rs. Blockade of Ca2+-induced Ca2+ release by TMB-8 following α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca2+ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca2+ overload whereas IP3R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by α subunit of the eukaryotic initiation factor 2α phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca2+ release through RyRs contributes to cytosolic Ca2+ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes. 相似文献
15.
Watarai H Sekine-Kondo E Shigeura T Motomura Y Yasuda T Satoh R Yoshida H Kubo M Kawamoto H Koseki H Taniguchi M 《PLoS biology》2012,10(2):e1001255
There is heterogeneity in invariant natural killer T (iNKT) cells based on the expression of CD4 and the IL-17 receptor B (IL-17RB), a receptor for IL-25 which is a key factor in TH2 immunity. However, the development pathway and precise function of these iNKT cell subtypes remain unknown. IL-17RB+
iNKT cells are present in the thymic CD44+/− NK1.1− population and develop normally even in the absence of IL-15, which is required for maturation and homeostasis of IL-17RB−
iNKT cells producing IFN-γ. These results suggest that iNKT cells contain at least two subtypes, IL-17RB+ and IL-17RB− subsets. The IL-17RB+
iNKT subtypes can be further divided into two subtypes on the basis of CD4 expression both in the thymus and in the periphery. CD4+ IL-17RB+
iNKT cells produce TH2 (IL-13), TH9 (IL-9 and IL-10), and TH17 (IL-17A and IL-22) cytokines in response to IL-25 in an E4BP4-dependent fashion, whereas CD4− IL-17RB+
iNKT cells are a retinoic acid receptor-related orphan receptor (ROR)γt+ subset producing TH17 cytokines upon stimulation with IL-23 in an E4BP4-independent fashion. These IL-17RB+
iNKT cell subtypes are abundantly present in the lung in the steady state and mediate the pathogenesis in virus-induced airway hyperreactivity (AHR). In this study we demonstrated that the IL-17RB+
iNKT cell subsets develop distinct from classical iNKT cell developmental stages in the thymus and play important roles in the pathogenesis of airway diseases. 相似文献
16.
Lanteri MC Kaidarova Z Peterson T Cate S Custer B Wu S Agapova M Law JP Bielawny T Plummer F Tobler LH Loeb M Busch MP Bramson J Luo M Norris PJ 《PloS one》2011,6(8):e22948
Background
West Nile virus (WNV) infection is asymptomatic in most individuals, with a minority developing symptoms ranging from WNV fever to serious neuroinvasive disease. This study investigated the impact of host HLA on the outcome of WNV disease.Methods
A cohort of 210 non-Hispanic mostly white WNV+ subjects from Canada and the U.S. were typed for HLA-A, B, C, DP, DQ, and DR. The study subjects were divided into three WNV infection outcome groups: asymptomatic (AS), symptomatic (S), and neuroinvasive disease (ND). Allele frequency distribution was compared pair-wise between the AS, S, and ND groups using χ2 and Fisher''s exact tests and P values were corrected for multiple comparisons (Pc). Allele frequencies were compared between the groups and the North American population (NA) used as a control group. Logistic regression analysis was used to evaluate the potential synergistic effect of age and HLA allele phenotype on disease outcome.Results
The alleles HLA-A*68, C*08 and DQB*05 were more frequently associated with severe outcomes (ND vs. AS, P A*68 = 0.013/Pc = 0.26, P C*08 = 0.0075/Pc = 0.064, and P DQB1*05 = 0.029/Pc = 0.68), However the apparent DQB1*05 association was driven by age. The alleles HLA-B*40 and C*03 were more frequently associated with asymptomatic outcome (AS vs. S, P B*40 = 0.021/Pc = 0.58 and AS vs. ND P C*03 = 0.039/Pc = 0.64) and their frequencies were lower within WNV+ subjects with neuroinvasive disease than within the North American population (NA vs. S, P B*40 = 0.029 and NA vs. ND, P C*03 = 0.032).Conclusions
Host HLA may be associated with the outcome of WNV disease; HLA-A*68 and C*08 might function as “susceptible” alleles, whereas HLA-B*40 and C*03 might function as “protective” alleles. 相似文献17.
A Steep Dependence of Inward-Rectifying Potassium Channels on
Cytosolic Free Calcium Concentration Increase Evoked by
Hyperpolarization in Guard Cells
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Inactivation of inward-rectifying
K+ channels (IK,in) by a rise in
cytosolic free [Ca2+] ([Ca2+]i)
is a key event leading to solute loss from guard cells and stomatal
closure. However, [Ca2+]i action on
IK,in has never been quantified, nor are its
origins well understood. We used membrane voltage to manipulate
[Ca2+]i (A. Grabov and M.R. Blatt [1998]
Proc Natl Acad Sci USA 95: 4778–4783) while recording
IK,in under a voltage clamp and
[Ca2+]i by Fura-2 fluorescence
ratiophotometry. IK,in inactivation
correlated positively with [Ca2+]i and
indicated a Ki of 329 ± 31
nm with cooperative binding of four Ca2+ ions
per channel. IK,in was promoted by the
Ca2+ channel antagonists Gd3+ and calcicludine,
both of which suppressed the [Ca2+]i rise,
but the [Ca2+]i rise was unaffected by the
K+ channel blocker Cs+. We also found that
ryanodine, an antagonist of intracellular Ca2+ channels
that mediate Ca2+-induced Ca2+ release, blocked
the [Ca2+]i rise, and Mn2+
quenching of Fura-2 fluorescence showed that membrane hyperpolarization
triggered divalent release from intracellular stores. These and
additional results point to a high signal gain in
[Ca2+]i control of
IK,in and to roles for discrete
Ca2+ flux pathways in feedback control of the
K+ channels by membrane voltage.Ca2+ underlies many fundamental regulatory processes
in plants, including adaptive responses to abiotic environmental stress
(Knight et al., 1996; Russell et al., 1996; McAinsh et al., 1997) and
programmed cell death evoked by pathogen attack (Low and Merida, 1996;
Hammondkosack and Jones, 1997). Coordination of changes in
[Ca2+]i and its
integration with downstream response elements are central in coupling
stimulus input to cellular response in these processes.In stomatal guard cells, the best characterized higher-plant cell
model, major downstream targets of
[Ca2+]i and their roles
in stomatal function have been identified. Increasing
[Ca2+]i is known to
inactivate IK,in and to activate
Cl− channels, events that bias plasma membrane
transport for net efflux of osmotically active solute and a loss of
turgor, which drives stomatal closure (Blatt and Grabov, 1997).
Furthermore, changes in
[Ca2+]i are associated
with ABA, CO2, and the growth hormone auxin
(Blatt and Grabov, 1997; McAinsh et al., 1997). These
[Ca2+]i signals have been
observed to oscillate (McAinsh et al., 1995; Webb et al., 1996),
characteristics that may constitute “Ca2+
signatures” to encode specific downstream responses (Berridge, 1996).
Yet, despite the evidence for
[Ca2+]i signaling in
guard cells, surprisingly little detail is known about the link between
[Ca2+]i changes and ion
channel activity at the plasma membrane or about the mechanisms
mediating such [Ca2+]i
changes. To our knowledge, in no instance have the characteristics of
ion channel regulation by Ca2+ been quantified
directly in any higher-plant cell.We recently described the coupling of membrane voltage to
[Ca2+]i, demonstrating
that hyperpolarization, whether under a voltage clamp or in the
presence of low [K+]o,
evoked [Ca2+]i increases
in guard cells, and that the voltage threshold for
[Ca2+]i rise was
profoundly altered by ABA (Grabov and Blatt, 1998). Our observations
indicated a link to Ca2+ influx across the plasma
membrane and raised questions about the efficacy of
[Ca2+]i in inactivating
IK,in and about the contributions of
intracellular Ca2+ release to the
[Ca2+]i signal. We have
used membrane voltage to experimentally manipulate
[Ca2+]i and report that
IK,in is strongly dependent on
[Ca2+]i, consistent with
a cooperative binding of four Ca2+ ions to effect
inactivation. Additional experiments indicate that voltage-evoked
[Ca2+]i increases depend
both on Ca2+ influx and on release of
Ca2+ from intracellular stores. These results
underscore the role of
[Ca2+]i as a high-gain
“switch” in the control of IK,in, and
implicate [Ca2+]i in
feedback control linking membrane voltage to the activity of the
K+ channels. 相似文献
18.
Xiong W Liu T Wang Y Chen X Sun L Guo N Zheng H Zheng L Ruat M Han W Zhang CX Zhou Z 《PloS one》2011,6(10):e24573
Aim
Neurotransmitter release is elicited by an elevation of intracellular Ca2+ concentration ([Ca2+]i). The action potential triggers Ca2+ influx through Ca2+ channels which causes local changes of [Ca2+]i for vesicle release. However, any direct role of extracellular Ca2+ (besides Ca2+ influx) on Ca2+-dependent exocytosis remains elusive. Here we set out to investigate this possibility on rat dorsal root ganglion (DRG) neurons and chromaffin cells, widely used models for studying vesicle exocytosis.Results
Using photolysis of caged Ca2+ and caffeine-induced release of stored Ca2+, we found that extracellular Ca2+ inhibited exocytosis following moderate [Ca2+]i rises (2–3 µM). The IC50 for extracellular Ca2+ inhibition of exocytosis (ECIE) was 1.38 mM and a physiological reduction (∼30%) of extracellular Ca2+ concentration ([Ca2+]o) significantly increased the evoked exocytosis. At the single vesicle level, quantal size and release frequency were also altered by physiological [Ca2+]o. The calcimimetics Mg2+, Cd2+, G418, and neomycin all inhibited exocytosis. The extracellular Ca2+-sensing receptor (CaSR) was not involved because specific drugs and knockdown of CaSR in DRG neurons did not affect ECIE.Conclusion/Significance
As an extension of the classic Ca2+ hypothesis of synaptic release, physiological levels of extracellular Ca2+ play dual roles in evoked exocytosis by providing a source of Ca2+ influx, and by directly regulating quantal size and release probability in neuronal cells. 相似文献19.
Background
Activated platelets exert a pro-inflammatory action that can be largely ascribed to their ability to interact with leukocytes and modulate their activity. We hypothesized that platelet activation and consequent formation of monocyte-platelet aggregates (MPA) induces a pro-inflammatory phenotype in circulating monocytes.Methodology/Principal Findings
CD62P+ platelets and MPA were measured, and monocytes characterized, by whole blood flow cytometry in healthy subjects, before and two days after receiving influenza immunization. Three monocytic subsets were identified: CD14+CD16−, CD14highCD16+and CD14lowCD16+. The increase in high sensitivity C-reactive protein post-immunization was accompanied by increased platelet activation and MPA formation (25.02±12.57 vs 41.48±16.81; p = 0.01), along with enhancement of circulating CD14highCD16+ cells (4.7±3.6 vs 10.4±4.8; p = 0.003), their percentage being linearly related to levels of CD62P+-platelets (r2 = 0.4347; p = 0.0008). In separate in vitro experiments, co-incubation of CD14+CD16− cells, isolated from healthy donor subjects, with autologous platelets gave rise to up-regulation of CD16 on monocytes as compared with those maintained in medium alone (% change in CD14+CD16+ cells following 48 h co-incubation of monocytes with platelets was +106±51% vs monocytes in medium alone; p<0.001). This effect correlated directly with degree of MPA formation (r2 = 0.7731; p<0.0001) and was associated with increased monocyte adhesion to endothelial cells. P-selectin glycoprotein ligand-1 (PSGL-1) blocking antibody, which abrogates MPA formation, abolished these effects, as did the cyclooxygenase (COX)-2 selective inhibitor NS-398, aspirin and the EP1/EP2-selective antagonist AH6809.Conclusions/Significance
These data suggest that MPA formation, as occurs in the blood under pro-inflammatory conditions, expands the pool of circulating CD14highCD16+ monocytes in a COX-2 dependent manner, and these monocytes exhibit increased adhesion to endothelium. Our findings delineate a novel mechanism underlying the pro-inflammatory effect of platelet activation. 相似文献20.
Regulation of PI(3)K/Akt signalling and cellular transformation by inositol polyphosphate 4‐phosphatase‐1
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Ivan Ivetac Rajendra Gurung Sandra Hakim Kristy A Horan David A Sheffield Lauren C Binge Philip W Majerus Tony Tiganis Christina A Mitchell 《EMBO reports》2009,10(5):487-493
Akt is a crucial phosphoinositide 3-kinase (PI(3)K) effector that regulates cell proliferation and survival. PI(3)K-generated signals, PtdIns(3,4,5)P3 and PtdIns(3,4)P2, direct Akt plasma membrane engagement. Pathological Akt plasma membrane association promotes oncogenesis. PtdIns(3,4)P2 is degraded by inositol polyphosphate 4-phosphatase-1 (4-ptase-1) forming PtdIns(3)P; however, the role of 4-ptase-1 in regulating the activation and function of Akt is unclear. In mouse embryonic fibroblasts lacking 4-ptase-1 (−/−MEFs), the Akt-pleckstrin homology (PH) domain was constitutively membrane-associated both in serum-starved and agonist-stimulated cells, in contrast to +/+MEFs, in which it was detected only at the plasma membrane following serum stimulation. Epidermal growth factor (EGF) stimulation resulted in increased Ser473 and Thr308-Akt phosphorylation and activation of Akt-dependent signalling in −/−MEFs, relative to +/+MEFs. Significantly, loss of 4-ptase-1 resulted in increased cell proliferation and decreased apoptosis. SV40-transformed −/−MEFs showed increased anchorage-independent cell growth and formed tumours in nude mice. This study provides the first evidence, to our knowledge, that 4-ptase-1 controls the activation of Akt and thereby cell proliferation, survival and tumorigenesis. 相似文献