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1.
V. L. Sharygin M. K. Pulatova T. G. Shlyakova I. N. Todorov Yu. I. Mitrokhin A. M. Wasserman N. D. Yordanov 《Biophysics》2009,54(2):214-222
The responses of dNTP, DNA, and protein synthesis systems in blood-forming organs of animals (dogs, mice) as well as changes in blood Fe3+-transferrin (Fe-TF) and Cu2+-ceruloplasmin (Cu-CP) pools upon γ-irradiation and administration of radioprotectors have been studied. It is shown that changes in Fe-TF and Cu-CP pools are indices of change in body radioresistance and are reliably checked by the EPR technique. An increase in the Fe-TF pool promotes the activation of synthesis of dNTP, DNA, and Fe3+-containing proteins, which are essential for repair efficiency during early postirradiation time as well as for the development of compensatory-restorative reactions of cellular systems; i.e., they are responsible for body resistance to DNA-damaging factors. It is important that the intensity of responses depends on the initial state of the organism. Thus, dogs with initial individual characteristics of blood typical of “depressed” or “activated” states had abnormally high responses to irradiation at low doses of 0.25 and 0.5 Gy. This fact is important for estimating the consequences of prolonged low-dose irradiation for the human population. It has been shown that radioprotectors efficient in the survival test activate the synthesis of dNTP, DNA, and proteins in organs. The intensity of dNTP synthesis and the time when dNTP pools become maximal determine the efficiency of protectors and the time of irradiation after their administration. 相似文献
2.
Pulatova MK Sharygin VL Shliakova TG 《Radiatsionnaia biologiia, radioecologiia / Rossi?skaia akademiia nauk》2003,43(1):29-43
The paper covers the problem on reactions of deoxyribonucleotide (dNTP) synthesis system in blood-forming organs of animals induced by irradiation. The synthesis of dNTP is a rate-limiting stage for DNA synthesis. Cellular requirements for dNTP pools during DNA synthesis are related with ensuring of the accuracy of DNA copying during replication and repair. It has been shown that organism defence mechanisms against irradiation include the following stages: 1. The prompt SOS-activation of dNTP synthesis 30 min later after irradiation, playing the important role in protecting of cell's genetic apparatus from damage. 2. The inhibition of dNTP synthesis within 3-24 h after irradiation resulting to the imbalance of four dNTP and the decrease of their pools. As result of that, the abnormal repair is observed due to depurinations, errors of base incorporations and "misrepair". 3. The restore of dNTP synthesis occurred 2 days later after irradiation. The increase of dNTP pools promotes the increase of DNA synthesis rate as well as proliferative activity of cells. Confirming the fact that the alterations in dNTP pools play essential role in the production of DNA lesions became an important step in understanding of the multistage process leading to radioprotection. To get high and balanced pools of dNTP needed for the increase in the volume of repair of DNA lesions the radioprotectors with high efficiency relative to the survival test were used in experiments. They induced the elevated dNTP synthesis in bone marrow and spleen during the time when the irradiation alone caused the essential prolonged suppression of dNTP synthesis as well as DNA and protein synthesis in organs of nonprotected animals. It has been shown that substances with antioxidant and antiradical activity induced the dNTP synthesis, too. In vivo regulatory factors of dNTP synthesis have been studied to elucidate the mechanisms of getting of high and balanced dNTP pools by using of different substances. 相似文献
3.
Cellular requirements for deoxyribonucleotide (dNTP) pools during DNA synthesis are related to ensuring of the accuracy of DNA copying during replication and repair. This paper covers some problems on the reactions of dNTP synthesis system in organs of animals against the treatment with DNA-damaging agents. Ribonucleoside diphosphate reductase (NDPR) is the key enzyme for the synthesis of dNTP, since it catalyses the reductive conversion of ribonucleotides to deoxyribonucleotides. The results obtained show that the rapid and transient increase in NDPR activity in animal organs occurs as cellular response against the treatment with DNA-damaging agents (SOS-type activation). We have also found the intensive radioprotector-stimulated activation of deoxyribonucleotide synthesis as well as DNA and protein synthesis in mice organs within 3 days after the administration of two radioprotectors, indralin and indometaphen, that provide the high animal survival. Our studies suggest that these effects are the most important steps in the protective mechanism of the radioprotectors and are responsible for the high animal survival. 相似文献
4.
Time-dependent responses of cellular systems in rat organs and Fe(3+)-transferrin and Cu(2+)-ceruloplasmin pools in blood to the blocking of translation by sublethal doses of cycloheximide (CHI) was studied by EPR spectroscopy and radioisotope techniques. It was shown that, within the early post-CHI-treatment time, the suppression of deoxyribonucleotide and DNA biosynthesis, the activation of catabolic enzymes, the inhibition of electron transfer in the mitochondrial electron transport chain, the activation and the following inactivation of cytochrome P-450, and an intensive production of nitrosyl complexes in rat blood and organs occur. In addition, the activation of the synthesis of steroid hormones in adrenal gland was revealed within 1-24 h after cycloheximide injection. In response to these metabolic disturbances, nonspecific compensatory recovery reactions developed, first of all, the "reprograming" of the translation process to produce new protein-synthesizing elements instead of cycloheximide-blocked ones. The activation of protein synthesis promotes the recovery of deoxyribonucleotide and DNA synthesis, the restoration of the redox state of mitochondrial and microsomal electron transport chains in organs as well as an increase of Fe(3+)-transferrin and Cu(2+)-ceruloplasmin pools in rat blood. These metabolic processes result in the full recovery of the functional ability of organs. 相似文献
5.
V. L. Sharygin M. K. Pulatova T. G. Shlyakova Yu. I. Mitrokhin I. N. Todorov 《Biology Bulletin》2005,32(4):331-349
The responses of the systems of synthesis of deoxyribonucleotides (dNTPs), DNA, and proteins in hematopoietic organs and liver of animals to γ-radiation, administration of radioprotectants and antioxidants as well as the dependence of these responses on the doses of radiation and drugs were studied. Radioprotectants of acute (indralin) and durable effects (indomethaphen) as well as natural α2-tocopherol) and synthetic antioxidants (ionol or 2,6-di-tert-butyl-4-methylphenol) efficient in survival test were used. Three stages could be recognized in the standard unspecific response of the studied systems to radiation: (1) immediate increase in ribonucleotide reductase activity in the tissues within the first 30 min as a part of the integrated SOS response to DNA damage, which activates dNTP synthesis; (2) inhibition of the synthesis of dNTPs, DNA, and proteins; and (3) restoring ribonucleotide reductase activity and integral increase in the production of dNTPs, DNA, and total protein, which is essential for the development of compensatory and restorative responses of the organism. The radioprotectants significantly increased ribonucleotide reductase activity, which increased intracellular concentrations of the four dNTP types in organs during radiation exposure and three following days. Within this period, ribonucleotide reductase activity was inhibited by 40–50% in animals not treated with radioprotectants as compared to control. Balanced high pools of dNTPs in the organs of radioprotectant-treated animals provided for high-performance repair of DNA damage. The radioprotectant-induced activation of dNTP synthesis during the development of compensatory and restorative responses provides for an earlier restoration of the cellular composition and functioning of the organs. Antioxidants stimulated the synthesis of dNTPs, DNA, and proteins in animal tissues in a strict dose interval. Their effect on the studied syntheses was dose-dependent: single or multiple long-term administration of high antioxidant doses inhibited synthesis of dNTPs, DNA, and proteins. Radioprotectants and antioxidants affected the pool of blood protein Fe3+-transferrin controlling the synthesis of iron-containing ribonucleotide reductase activity in hematopoietic organs, and hence, the iron-dependent stage in DNA synthesis—dNTP synthesis. Activation of protein synthesis in organs by the studied substances increased the pools of Fe3+-transferrin and Cu2+-ceruloplasmin in the blood, which activated dNTP and DNA synthesis. Activated synthesis of dNTP, DNA, and proteins in the organs and increased pools of studied plasma proteins underlay the formation of body resistance to DNA-damaging factors.__________Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 4, 2005, pp. 401–422.Original Russian Text Copyright © 2005 by Sharygin, Pulatova, Shlyakova, Mitrokhin, Todorov. 相似文献
6.
We investigated the combined effects of low-energy electron irradiation and Fe(3+) ion on DNA damage. We used lyophilized pBR322 plasmid DNA films with various concentrations (0 ~ 7 mM) of Fe(3+) ions and irradiation with monochromatic, low-energy 3 or 5 eV electrons for these studies. DNA-Fe(3+) films were recovered and analyzed by agarose gel electrophoresis to identify and compare the effects of Fe(3+) ions and/or low-energy electrons alone or in combination on DNA damage. In nonirradiated DNA-Fe(3+) films, there was little DNA damage observed (less than 10% of the total DNA loaded on the gel appeared damaged) for Fe(3+) ion up to 7 mM concentration. In irradiated DNA films without Fe(3+) ions, there was also very little DNA damage observed (less than 3% of the total DNA loaded on the gel appeared damaged). However, when DNA-Fe(3+) films, were irradiated with low-energy electrons, DNA damage was significantly increased compared to the sum of the damage caused both by either Fe(3+) ion or low-energy electrons irradiation alone. We proposed that both DEA and/or electron transfer processes might play a role in the enhanced DNA damage when DNA-Fe(3+) films were irradiated by low-energy electrons. 相似文献
7.
Kinetics of iron release from ferric binding protein (FbpA): mechanistic implications in bacterial periplasm-to-cytosol Fe3+ transport 总被引:1,自引:0,他引:1
The ferric binding protein (FbpA) transports iron across the periplasmic space of certain Gram-negative bacteria and is an important component involved in iron acquisition by pathogenic Neisseria spp. (Neisseria gonorrheae and Neisseria meningitidis). Previous work has demonstrated that the synergistic anion, required for tight Fe(3+) sequestration by FbpA, also plays a key role in inserting Fe(3+) into the FbpA binding site. Here, we investigate the iron release process from various forms of holo-FbpA, Fe(3+)FbpA-X, during the course of a chelator competition reaction using EDTA and Tiron. Fe(3+)FbpA-X represents the protein assembly complex with different synergistic anions, X = PO(4)(3)(-) and NTA. Stepwise mechanisms of Fe(3+) release are proposed on the basis of kinetic profiles of these chelator competition reactions. Fe(3+)FbpA-PO(4) and Fe(3+)FbpA-NTA react differently with EDTA and Tiron during the Fe(3+)-exchange process. EDTA replaces PO(4)(3)(-) and NTA from the first coordination shell of Fe(3+) and acts as a synergistic anion to give a spectroscopically distinguishable intermediate, Fe(3+)FbpA-EDTA, prior to pulling Fe(3+) out of the protein. Tiron, on the other hand, does not act as a synergistic anion but is a more efficient competing chelator as it removes Fe(3+) from FbpA at rate much faster than EDTA. These results reaffirm the contribution of the synergistic anion to the FbpA iron transport process as the anion, in addition to playing a facilitative role in iron binding, appears to have a "gatekeeper" role, thereby modulating the Fe(3+) release process. 相似文献
8.
M K Pulatova D E Filatov T G Shliakova V L Sharygin V P Beletski? G A Chernov 《Izvestiia Akademii nauk SSSR. Seriia biologicheskaia》1992,(2):215-233
The study deals with the mechanism of organism's adaptive responses to the effect of radiation in widely ranging dose. Post-irradiation metabolic changes were evaluated in canine blood as well as in murine blood, spleen, bone marrow and liver using the EPR spectroscopy. It was shown that the dynamics of changes in transferrin and ceruloplasmin pools and ribonucleotide reductase activity were phase-dependent with the maxima at the 2nd, 6th and 10-12th days after irradiation. Such dynamics was observed at various irradiation doses applied. The data allow us to suggest that the nonspecific compensatory--adaptive reactions of organisms develop as the response to irradiation. The dose-response function of the reaction intensity was found to be linear. The shape of the dose-response curve indicates that the minimum response of organism depends on the dose linearly up to 3.2 Gy (for dogs) as well as the maximum one. However, in the case of low-dose irradiation (0.25 or 0.5 Gy) there were deviations of maximum responses from the linearity, i.e. the amplification of the amplitude of compensatory adaptive reactions. These effect were shown to be dependent upon initial individual characteristics of animal blood and to be related to the "depressed" or "activated" state of organism prior to irradiation. The ribonucleotide reductase activity was measured in bone marrow and spleen of animals by the EPR method. The nature of non-repairable DNA damage is discussed in view of the inactivation of ribonucleotide reductase. 相似文献
9.
A A Pirogov A I Erofeev A P Kozlov 《Zhurnal vysshe? nervno? deiatelnosti imeni I P Pavlova》1990,40(2):331-339
From 192 neurons, recorded in the frontal cortex of dogs trained for differential classical conditioning of salivary reflex, 24 cells were identified related to the detection of matching-mismatching conditioned and unconditioned stimuli (CS and US) to internal set. Impulse reactions of the 1-st group of neurones (11 cells) to US disappeared, when any deviation from standard pattern of CS-US pairing occurred or when US characteristics were suddenly changed. These reactions developed again at the 3-5th repeated presentation of the stimuli delivered in "new" regime. Neurones of the 2-nd group (13 cells) were activated in response to CS only when two sequential CSs had different meaning. The 1-st group of neurones is considered as detectors of matching, and the 2-nd group--as detectors of mismatching the stimulus to its neural model, formed by mechanism of reinforcement. 相似文献
10.
E N Goncharenko E E Graevskaia T G Gorskaia N N Lomakin 《Nauchnye doklady vysshe? shkoly. Biologicheskie nauki》1985,(3):28-30
It has been shown that the process of amines new formation in mast cells after the treatment with radioprotectors is less intensive than in other tissues able to synthesize amines. It is proposed that the effect of the mast cells in organism's formation of higher radioresistance is brought to mobilization of the accumulated biogenic amines with their further redistribution in other organs and tissues including radiosensitive ones. 相似文献
11.
Crouzin N de Jesus Ferreira MC Cohen-Solal C Aimar RF Vignes M Guiramand J 《Free radical biology & medicine》2007,42(9):1326-1337
We have reported that a transient treatment of hippocampal neurons with alpha-tocopherol induced a long-lasting protection against oxidative damage mediated by Fe(2+) ions. This protection required protein synthesis. Here, we have studied whether this "hyposensitivity" to oxidative stress could be linked to an altered Ca(2+) homeostasis. Fe(2+) ions triggered a Ca(2+) entry which was required for Fe(2+) ion-induced toxicity. This influx was sensitive to blockers of TRP-like nonspecific Ca(2+) channels, including Ruthenium Red, La(3+), and Gd(3+) ions which also prevented the Fe(2+) ion-induced toxicity and oxidative stress as revealed by protein carbonylation status. The pretreatment with alpha-tocopherol resulted in a reduction of the Ca(2+) increase induced by Fe(2+) ions and masked the blocking effect of La(3+) ions. Moreover, such a pretreatment reduced the capacitive Ca(2+) entries (CCE) observed after metabotropic glutamate receptor stimulation, which are known to involve TRP-like channels. By contrast, in a model of "hypersensitivity" to oxidative stress obtained by chronic stimulation of glucocorticoid receptors, we observed an exacerbation of the various effects of Fe(2+) ions, i.e., cellular toxicity and Ca(2+) increase, and the glutamate-stimulated CCE. Therefore, we conclude that the long-lasting neuroprotection induced by alpha-tocopherol pretreatment likely results from an attenuation of Ca(2+) entries via TRP-like channels. 相似文献
12.
N Macková P Fedorocko P Brezáni 《Physiological research / Academia Scientiarum Bohemoslovaca》1991,40(5):503-512
The culture medium administered to C57Bl/6 mice 18 h and 8 h before a single irradiation (9 Gy) had a radioprotective effect and clearly influenced postirradiation changes in haemopoiesis. Haemopoiesis recovery appeared to be faster in culture medium-pretreated animals than in those irradiated without such pretreatment. By 12-15 days after irradiation, the thymus cortex appeared to be repaired, on day 21 a multiple increase in extramedullary erythropoiesis, myelopoiesis and megakaryocytopoiesis in the red pulp was found and later, on day 28, the lymphopoiesis in the white pulp of spleen was restored. The rate of haemopoiesis proliferation of predominantly myeloid cells which reached a control level on day 28 following irradiation. Consequently, the regenerative processes in blood-forming organs were accompanied by considerable reticulocytosis and complete recovery of neutrophil and platelet counts in the peripheral blood as seen on day 21. Despite a slower rate complete recovery of the total leukocyte count was reached by day 180 after irradiation. 相似文献
13.
DNA precursor pools and ribonucleotide reductase activity: distribution between the nucleus and cytoplasm of mammalian cells. 总被引:13,自引:3,他引:10
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Nuclear and whole-cell deoxynucleoside triphosphate (dNTP) pools were measured in HeLa cells at different densities and throughout the cell cycle of synchronized CHO cells. Nuclei were prepared by brief detergent (Nonidet P-40) treatment of subconfluent monolayers, a procedure that solubilizes plasma membranes but leaves nuclei intact and attached to the plastic substratum. Electron microscopic examination of monolayers treated with Nonidet P-40 revealed protruding nuclei surrounded by cytoskeletal remnants. Control experiments showed that nuclear dNTP pool sizes were stable during the time required for isolation, suggesting that redistribution of nucleotides during the isolation procedure was minimal. Examination of HeLa whole-cell and nuclear dNTP levels revealed that the nuclear proportion of each dNTP was distinct and remained constant as cell density increased. In synchronized CHO cells, all four dNTP whole-cell pools increased during S phase, with the dCTP pool size increasing most dramatically. The nuclear dCTP pool did not increase as much as the whole-cell dCTP pool during S phase, lowering the relative nuclear dCTP pool. Although the whole-cell dNTP pools decreased after 30 h of isoleucine deprivation, nuclear pools did not decrease proportionately. In summary, nuclear dNTP pools in synchronized CHO cells maintained a relatively constant concentration throughout the cell cycle in the face of larger fluctuations in whole-cell dNTP pools. Ribonucleotide reductase activity was measured in CHO cells throughout the cell cycle, and although there was a 10-fold increase in whole-cell activity during S phase, we detected no reductase in nuclear preparations at any point in the cell cycle. 相似文献
14.
Yamaoka K Mori S Nomura T Taguchi T Ito T Hanamoto K Kojima S 《Physiological chemistry and physics and medical NMR》2002,34(2):119-132
The increase in lipid peroxide levels in mice brain following Fe3+ administration was about 50% of that when 1-methyl-4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) was administered. This may be due to excessive oxidation by Fe3+, and was supported by the decrease in activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase and glutathione peroxidase (GPX), Na+,K(+)-ATPase activity and membrane fluidity after Fe3+ administration. Relatively low-dose X-ray irradiation (0.5 Gy) inhibited lipid peroxidation associated with Fe3+ administration and restored the decreased activities of the above antioxidant enzymes and Na+,K(+)-ATPase, and membrane fluidity to the levels in the non-Fe(3+)-administered group. In the purine metabolism system, uric acid decreased after Fe3+ administration, which may be due to transient impairment of the system for production of uric acid from xanthine by excessive oxidation by Fe3+. However, 0.5 Gy irradiation inhibited this decrease in uric acid, increasing its level to that in the non Fe(3+)-administrated group. This may be due to factors such as rapid recovery of the activities of the above antioxidant enzymes and Na+,K(+)-ATPase, and membrane fluidity after 0.5 Gy irradiation. In addition, since no changes were observed in xanthine and uric acid, increased inosine and hypoxanthine may have advanced to a salvage pathway leading to not xanthine but inosine 5'-monophosphate (IMP). 相似文献
15.
The effect of UV irradiation on the intracellular DNA precursor pool in E. coli was investigated. UV irradiation of E. coli, followed by post-incubation for 1-1.5 h, altered the relative sizes of the deoxyribonucleoside triphosphate (dNTP) pool. The total amount of dNTPs increased: both dATP and dTTP increased several-fold, dCTP about twofold, while dGTP remained almost unchanged. In recA- and umuC- strains, which are defective in UV-induced mutagenesis, the pattern of nucleotide pool alterations was similar to that of wild-type strains. 相似文献
16.
Intracellular pool sizes of deoxyribonucleoside triphosphates (dNTPs) are highly regulated. Unbalanced dNTP pools, created by abnormal accumulation or deficiency of one nucleotide, are known to be mutagenic and to have other genotoxic consequences. Recent studies in our laboratory on DNA replication in vitro suggested that balanced accumulation of dNTPs, in which all four pools increase proportionately, also stimulates mutagenesis. In this paper, we ask whether proportional dNTP pool increases are mutagenic also in living cells. Escherichia coli was transformed with recombinant plasmids that overexpress E. coli genes nrdA and nrdB, which encode the two protein subunits of aerobic ribonucleotide reductase. Roughly proportional dNTP pool expansion, by factors of 2- to 6-fold in different experiments, was accompanied by increases in spontaneous mutation frequency of up to 40-fold. Expression of a catalytically inactive ribonucleotide reductase had no effect on either dNTP pools or mutagenesis, suggesting that accumulation of dNTPs is responsible for the increased mutagenesis. Preliminary experiments with strains defective in SOS regulon induction suggest a requirement for one or more SOS functions in the dNTP-enhanced mutagenesis. Because a replisome extending from correctly matched 3'-terminal nucleotides is almost certainly saturated with dNTP substrates in vivo, whereas chain extension from mismatched nucleotides almost certainly proceeds at sub-saturating rates, we propose that the mutagenic effect of proportional dNTP pool expansion is preferential stimulation of chain extension from mismatches as a result of increases in intracellular dNTP concentrations. 相似文献
17.
Mizutani K Muralidhara BK Yamashita H Tabata S Mikami B Hirose M 《The Journal of biological chemistry》2001,276(38):35940-35946
The differential properties of anion-mediated Fe(3+) release between the N- and C-lobes of transferrins have been a focus in transferrin biochemistry. The structural and kinetic characteristics for isolated lobe have, however, been documented with the N-lobe only. Here we demonstrate for the first time the quantitative Fe(3+) release kinetics and the anion-binding structure for the isolated C-lobe of ovotransferrin. In the presence of pyrophosphate, sulfate, and nitrilotriacetate anions, the C-lobe released Fe(3+) with a decelerated rate in a single exponential progress curve, and the observed first order rate constants displayed a hyperbolic profile as a function of the anion concentration. The profile was consistent with a newly derived single-pathway Fe(3+) release model in which the holo form is converted depending on the anion concentration into a "mixed ligand" intermediate that releases Fe(3+). The apo C-lobe was crystallized in ammonium sulfate solution, and the structure determined at 2.3 A resolution demonstrated the existence of a single bound SO(4)(2-) in the interdomain cleft, which interacts directly with Thr(461)-OG1, Tyr(431)-OH, and His(592)-NE2 and indirectly with Tyr(524)-OH. The latter three groups are Fe(3+)-coordinating ligands, strongly suggesting the facilitated Fe(3+) release upon the anion occupation at this site. The SO(4)(2-) binding structure supported the single-pathway kinetic model. 相似文献
18.
The characterization of iron handling in neurons is still lacking, with contradictory and incomplete results. In particular, the relevance of non-transferrin-bound iron (NTBI), under physiologic conditions, during aging and in neurodegenerative disorders, is undetermined. This study investigates the mechanisms underlying NTBI entry into primary hippocampal neurons and evaluates the consequence of iron elevation on neuronal viability. Fluorescence-based single cell analysis revealed that an increase in extracellular free Fe(2+) (the main component of NTBI pool) is sufficient to promote Fe(2+) entry and that activation of either N-methyl-d-aspartate receptors (NMDARs) or voltage operated calcium channels (VOCCs) significantly potentiates this pathway, independently of changes in intracellular Ca(2+) concentration ([Ca(2+) ](i) ). The enhancement of Fe(2+) influx was accompanied by a corresponding elevation of reactive oxygen species (ROS) production and higher susceptibility of neurons to death. Interestingly, iron vulnerability increased in aged cultures. Scavenging of mitochondrial ROS was the most powerful protective treatment against iron overload, being able to preserve the mitochondrial membrane potential and to safeguard the morphologic integrity of these organelles. Overall, we demonstrate for the first time that Fe(2+) and Ca(2+) compete for common routes (i.e. NMDARs and different types of VOCCs) to enter primary neurons. These iron entry pathways are not controlled by the intracellular iron level and can be harmful for neurons during aging and in conditions of elevated NTBI levels. Finally, our data draw the attention to mitochondria as a potential target for the treatment of the neurodegenerative processes induced by iron dysmetabolism. 相似文献
19.
Taboy CH Vaughan KG Mietzner TA Aisen P Crumbliss AL 《The Journal of biological chemistry》2001,276(4):2719-2724
The Fe(3+) binding site of recombinant nFbp, a ferric-binding protein found in the periplasmic space of pathogenic Neisseria, has been characterized by physicochemical techniques. An effective Fe(3+) binding constant in the presence of 350 microm phosphate at pH 6.5 and 25 degrees C was determined as 2.4 x 10(18) m(-1). EPR spectra for the recombinant Fe(3+)nFbp gave g' = 4.3 and 9 signals characteristic of high spin Fe(3+) in a strong ligand field of low (orthorhombic) symmetry. (31)P NMR experiments demonstrated the presence of bound phosphate in the holo form of nFbp and showed that phosphate can be dialyzed away in the absence of Fe(3+) in apo-nFbp. Finally, an uncorrected Fe(3+/2+) redox potential for Fe-nFbp was determined to be -290 mV (NHE) at pH 6.5, 20 degrees C. Whereas our findings show that nFbp and mammalian transferrin have similar Fe(3+) binding constants and EPR spectra, they differ greatly in their redox potentials. This has implications for the mechanism of Fe transport across the periplasmic space of Gram-negative bacteria. 相似文献
20.
Tran CH Taylor MS Plane F Nagaraja S Tsoukias NM Solodushko V Vigmond EJ Furstenhaupt T Brigdan M Welsh DG 《American journal of physiology. Cell physiology》2012,302(8):C1226-C1242
When arteries constrict to agonists, the endothelium inversely responds, attenuating the initial vasomotor response. The basis of this feedback mechanism remains uncertain, although past studies suggest a key role for myoendothelial communication in the signaling process. The present study examined whether second messenger flux through myoendothelial gap junctions initiates a negative-feedback response in hamster retractor muscle feed arteries. We specifically hypothesized that when agonists elicit depolarization and a rise in second messenger concentration, inositol trisphosphate (IP(3)) flux activates a discrete pool of IP(3) receptors (IP(3)Rs), elicits localized endothelial Ca(2+) transients, and activates downstream effectors to moderate constriction. With use of integrated experimental techniques, this study provided three sets of supporting observations. Beginning at the functional level, we showed that blocking intermediate-conductance Ca(2+)-activated K(+) channels (IK) and Ca(2+) mobilization from the endoplasmic reticulum (ER) enhanced the contractile/electrical responsiveness of feed arteries to phenylephrine. Next, structural analysis confirmed that endothelial projections make contact with the overlying smooth muscle. These projections retained membranous ER networks, and IP(3)Rs and IK channels localized in or near this structure. Finally, Ca(2+) imaging revealed that phenylephrine induced discrete endothelial Ca(2+) events through IP(3)R activation. These events were termed recruitable Ca(2+) wavelets on the basis of their spatiotemporal characteristics. From these findings, we conclude that IP(3) flux across myoendothelial gap junctions is sufficient to induce focal Ca(2+) release from IP(3)Rs and activate a discrete pool of IK channels within or near endothelial projections. The resulting hyperpolarization feeds back on smooth muscle to moderate agonist-induced depolarization and constriction. 相似文献