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991.
Hilgert M Hartmann J Löffelholz K Jeltsch H Cassel JC Klein J 《Neurochemical research》2003,28(3-4):467-472
The cholinergic inputs to the rat hippocampus were lesioned by intraseptal injections of 192 IgG-saporin. After 15 days, fetal septal cells were grafted into the hippocampus. Thirteen months later, hippocampal acetylcholine (ACh) release was studied by microdialysis. Lesioning reduced basal ACh release (100%) to 20% of normal, which was compensated for by the graft (71%). Infusion of the serotonin uptake inhibitor citalopram (100 M) enhanced ACh release to the same extent (% of basal release) in all rat groups. Systemic injection of 8-OH-DPAT (0.5 mg/kg, SC), an agonist of 5-HT1A receptors, caused a smaller ACh release than citalopram. Acetylcholinesterase (AChE) staining and densitometric quantification revealed that the lesion-induced reduction of the AChE-staining density was compensated for by septal grafting. In conclusion, both histochemical and biochemical methods showed that cholinergic hippocampal parameters were drastically impaired by 192 IgG-saporin lesions, but were almost completely restored by septal grafting. The graft responded to intrinsic serotonergic regulation. 相似文献
992.
Motions and structural variability within toxins: implication for their use as scaffolds for protein engineering 下载免费PDF全文
Gilquin B Bourgoin M Ménez R Le Du MH Servent D Zinn-Justin S Ménez A 《Protein science : a publication of the Protein Society》2003,12(2):266-277
Animal toxins are small proteins built on the basis of a few disulfide bonded frameworks. Because of their high variability in sequence and biologic function, these proteins are now used as templates for protein engineering. Here we report the extensive characterization of the structure and dynamics of two toxin folds, the "three-finger" fold and the short alpha/beta scorpion fold found in snake and scorpion venoms, respectively. These two folds have a very different architecture; the short alpha/beta scorpion fold is highly compact, whereas the "three-finger" fold is a beta structure presenting large flexible loops. First, the crystal structure of the snake toxin alpha was solved at 1.8-A resolution. Then, long molecular dynamics simulations (10 ns) in water boxes of the snake toxin alpha and the scorpion charybdotoxin were performed, starting either from the crystal or the solution structure. For both proteins, the crystal structure is stabilized by more hydrogen bonds than the solution structure, and the trajectory starting from the X-ray structure is more stable than the trajectory started from the NMR structure. The trajectories started from the X-ray structure are in agreement with the experimental NMR and X-ray data about the protein dynamics. Both proteins exhibit fast motions with an amplitude correlated to their secondary structure. In contrast, slower motions are essentially only observed in toxin alpha. The regions submitted to rare motions during the simulations are those that exhibit millisecond time-scale motions. Lastly, the structural variations within each fold family are described. The localization and the amplitude of these variations suggest that the regions presenting large-scale motions should be those tolerant to large insertions or deletions. 相似文献
993.
Genetic structure and gene flow in a metapopulation of an endangered plant species,Silene tatarica 总被引:1,自引:0,他引:1
We investigated the distribution of genetic variation within and between seven subpopulations in a riparian population of Silene tatarica in northern Finland by using amplified fragment length polymorphism (AFLP) markers. A Bayesian approach-based clustering program indicated that the marker data contained not only one panmictic population, but consisted of seven clusters, and that each original sample site seems to consist of a distinct subpopulation. A coalescent-based simulation approach shows recurrent gene flow between subpopulations. Relative high FST values indicated a clear subpopulation differentiation. However, amova analysis and UPGMA-dendrogram did not suggest any hierarchical regional structuring among the subpopulations. There was no correlation between geographical and genetic distances among the subpopulations, nor any correlation between the subpopulation census size and amount of genetic variation. Estimates of gene flow suggested a low level of gene flow between the subpopulations, and the assignment tests proposed a few long-distance bidirectional dispersal events between the subpopulations. No apparent difference was found in within-subpopulation genetic diversity among upper, middle and lower regions along the river. Relative high amounts of linkage disequilibrium at subpopulation level indicated recent population bottlenecks or admixture, and at metapopulation levels a high subpopulation turnover rate. The overall pattern of genetic variation within and between subpopulations also suggested a 'classical' metapopulation structure of the species suggested by the ecological surveys. 相似文献
994.
Screening for functional expression and overexpression of a family of diiron-containing interfacial membrane proteins using the univector recombination system 下载免费PDF全文
Berthold DA Stenmark P Nordlund P 《Protein science : a publication of the Protein Society》2003,12(1):124-134
The large number of uncharacterized genes emerging from genome sequencing projects has resulted in a need for quick and reliable screening methods for protein expression parameters. We have utilized the univector plasmid recombination system (as previously reported) to develop a series of vectors for rapid screening for expression in Escherichia coli. A high level of recombinant protein expression is a requirement for purification of protein for structural determination and other purposes. In other applications, successful complementation of a missing enzyme activity in E. coli, as well as directed evolution studies and metabolic engineering, often require a much lower level of protein expression. In this report we describe the construction of a number of new pHOST vectors that can be screened for both low- and high-level expression. We isolated a mutant vector for MBP fusions that exhibited a more optimal level of expression for complementation of aerobic respiration in hemA(-) E. coli, our functional assay for the alternative oxidase. We then demonstrated the use of our system to rapidly screen for both optimal functional expression and optimal overexpression of the alternative oxidase as well as two other members of a family of membrane-bound diiron carboxylate proteins, the plastid terminal oxidase and 5-demethoxyquinone hydroxylase. 相似文献
995.
Reidy MF Wright JR 《American journal of physiology. Lung cellular and molecular physiology》2003,285(4):L854-L861
The phagocytosis of apoptotic inflammatory cells by alveolar macrophages (AMs) is a key component of inflammation resolution within the air space. Surfactant protein A (SP-A) has been shown to stimulate the phagocytosis of apoptotic neutrophils (PMNs) by normal AMs. We hypothesized that SP-A promotes the resolution of alveolar inflammation by enhancing apoptotic PMN phagocytosis and anti-inflammatory cytokine release by inflammatory AMs. Using an LPS lung inflammation model, we determined that SP-A stimulates the phagocytosis of apoptotic PMNs threefold by normal AMs and AMs isolated after LPS injury. Furthermore, SP-A enhances transforming growth factor-beta1 (TGF-beta1) release from both AM populations. Inflammatory AMs release twofold more TGF-beta1 in culture than do normal AMs. SP-A and apoptotic PMNs together stimulate TGF-beta1 release equivalently from normal and inflammatory cultured AMs (330% of unstimulated release by normal AMs). In summary, SP-A enhances apoptotic PMN uptake, stimulates AM TGF-beta1 release, and modulates the amount of TGF-beta1 released when AMs phagocytose apoptotic PMNs. These findings support the hypothesis that SP-A promotes the resolution of alveolar inflammation. 相似文献
996.
Brinker KG Garner H Wright JR 《American journal of physiology. Lung cellular and molecular physiology》2003,284(1):L232-L241
Surfactant protein A (SP-A) is an innate immune molecule that regulates pathogen clearance and lung inflammation. SP-A modulates innate immune functions such as phagocytosis, cytokine production, and chemotaxis; however, little is known about regulation of adaptive immunity by SP-A. Dendritic cells (DCs) are the most potent antigen-presenting cell with the unique capacity to activate naive T cells and initiate adaptive immunity. The goal of this study was to test the hypothesis that SP-A regulates the differentiation of immature DCs into potent T cell stimulators. The data show that incubation of immature DCs for 24 h with SP-A inhibits basal- and LPS-mediated expression of major histocompatibility complex class II and CD86. Stimulation of immature DCs by SP-A also inhibits the allostimulation of T cells, enhances dextran endocytosis, and alters DC chemotaxis toward RANTES and secondary lymphoid tissue chemokine. The effects on DC phenotype and function are similar for the structurally homologous C1q, but not for SP-D. These studies demonstrate that SP-A participates in the adaptive immune response by modulating important immune functions of DCs. 相似文献
997.
Impaired secretion of IL-10 by T cells from patients with common variable immunodeficiency--involvement of protein kinase A type I 总被引:2,自引:0,他引:2
Holm AM Aukrust P Aandahl EM Müller F Taskén K Frøland SS 《Journal of immunology (Baltimore, Md. : 1950)》2003,170(11):5772-5777
Common variable immunodeficiency (CVID) is a heterogeneous group of B cell deficiency syndromes. T cell abnormalities are present in a high proportion of patients with CVID, suggesting impaired T cell-mediated stimulation of B cells. Based on the importance of IL-10 for B cell function and the involvement of the cAMP/protein kinase A type I (PKAI) system in IL-10 synthesis, we examined IL-10 secretion in T cells from CVID patients and controls, particularly focusing on possible modulatory effects of the cAMP/PKAI system. Our main findings were: 1) anti-CD3 and anti-CD3/anti-CD28 activated T cells from CVID patients secreted less IL-10 than healthy controls. This defect was not related to varying proportions of T cell subsets (e.g., CD4(+)/CD8(+), CD45RA(+)/RO(+), or CD28(-) T cells); 2) PKAI activation through the cAMP agonist 8-CPT-cAMP markedly inhibited IL-10 secretion from T cells through CD3 and CD28 activation in both patients and controls, but the sensitivity for cAMP-dependent inhibition was increased in CVID; 3) selective PKAI inhibition by Rp-8-Br-cAMPS markedly increased IL-10 secretion in anti-CD3 and anti-CD3/anti-CD28-stimulated T cells in both patients and controls. Even at the lowest concentrations of Rp-8-Br-cAMPS, IL-10 secretion in CVID patients reached levels comparable to those in controls. Our findings suggest impaired secretion of IL-10 by T cells from CVID patients, suggesting a possible link between T cell deficiency and impaired B cell function in CVID. The involvement of the cAMP/PKAI system in this defect suggests a novel target for therapeutic immunomodulation in CVID. 相似文献
998.
Rego AC Monteiro NM Silva AP Gil J Malva JO Oliveira CR 《Journal of neurochemistry》2003,86(4):792-804
In the present work we investigated the effect of selective stimulation of non-desensitizing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in the intracellular processes leading to hippocampal neuronal death and production of reactive oxygen species (ROS). Activation of AMPA receptors in the presence of cyclothiazide (CYZ), a blocker of AMPA receptor desensitization, resulted in the death of approximately 25% of neurones, which was prevented by 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(f)quinoxaline (NBQX), an AMPA-preferring receptor antagonist. (+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) protected the neurones from necrotic death induced by AMPA or NMDA receptor activation. Neurodegeneration caused by selective activation of non-desensitizing AMPA receptors, in the presence of AMPA, CYZ and MK-801, significantly decreased the number of Co2+-positive neurones, used as a cytochemical marker of Ca2+-permeable AMPA receptors, but maintained intracellular ATP/ADP. The AMPA-mediated apoptotic cell death involved mitochondrial cytochrome c release and the activation of caspases-1 and -3, which was prevented by NBQX. Interestingly, although selective activation of AMPA receptors was not associated with production of intracellular peroxides, a moderate increase in superoxide production was observed upon exposure to antimycin A (AA). Furthermore, increased activity of Mn- superoxide dismutase (SOD) was observed on selective activation of non-desensitizing AMPA receptors. Taken together, these data make important contributions to the elucidation of the downstream pathways activated in AMPA receptor-mediated excitotoxicity in cultured rat hippocampal neurones. 相似文献
999.
The myelin-associated glycoprotein (MAG) is a transmembrane cell adhesion molecule participating in myelin formation and maintenance. Calcium-activated/-dependent proteolysis of myelin-associated glycoprotein by calpain and cathepsin L-like activities has already been detected in purified myelin fractions, producing a soluble fragment, called degraded (d)MAG, characterized by the loss of the transmembrane and cytoplasmic domains. Here, we demonstrate and analyze dMAG formation from pure human brain myelin-associated glycoprotein. The activity never exhibited the high rate previously reported in human myelin fractions. Degradation is time-, temperature-, buffer- and structure-dependent, is inhibited at 4 degrees C and by denaturation of the sample, and is mediated by a trans-acting factor. There is no strict pH dependency of the proteolysis. Degradation was inhibited by excess aprotinin, but not by 1-10 micro g/mL aprotinin and was not eliminated by the use of an aprotinin-sepharose matrix during the purification. dMAG formation was not enhanced by calcium, nor inhibited by a wide variety of protease inhibitors, including specific calpain and cathepsin L inhibitors. Therefore, while cysteine proteases may be present in human myelin membrane fractions, they are not involved in dMAG formation from highly purified human brain myelin-associated glycoprotein preparations. 相似文献
1000.
The cytoskeleton is a dynamic three-dimensional structure mainly located in the cytoplasm. It is involved in many cell functions such as mechanical signal transduction and maintenance of cell integrity. Among the three cytoskeletal components, intermediate filaments (the cytokeratin in epithelial cells) are the best candidates for this mechanical role. A model of the establishment of the cytokeratin network of an epithelial cell is proposed to study the dependence of its structural organization on extracellular mechanical environment. To implicitly describe the latter and its effects on the intracellular domain, we use mechanically regulated protein synthesis. Our model is a hybrid of a partial differential equation of parabolic type, governing the evolution of the concentration of cytokeratin, and a set of stochastic differential equations describing the dynamics of filaments. Each filament is described by a stochastic differential equation that reflects both the local interactions with the environment and the non-local interactions via the past history of the filament. A three-dimensional simulation model is derived from this mathematical model. This simulation model is then used to obtain examples of cytokeratin network architectures under given mechanical conditions, and to study the influence of several parameters. 相似文献