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51.
The ability to visualize plasmid DNA entrapment in muscle cells undergoing an "in vivo" electroporation treatment was investigated on BALB/c mice using a 7-T magnetic resonance imaging (MRI) scanner using the paramagnetic Gd-DOTA-spd complex as imaging reporter. Gd-DOTA-spd bears a tripositively charged spermidine residue that yields a strong binding affinity toward the negatively charged DNA chain (6.4 kb, K(a) = 2.2 x 10(3) M(-1) for approximately 2500 +/- 500 binding sites). Cellular colocalization of Gd-DOTA-spd and plasmid DNA has been validated by histological analysis of excised treated muscle. In vivo MRI visualization of Gd-DOTA-spd distribution provides an excellent route to access the cellular entrapment of plasmid DNA upon applying an electroporation pulse. 相似文献
52.
Pedone E Saviano M Bartolucci S Rossi M Ausili A Scirè A Bertoli E Tanfani F 《Journal of proteome research》2005,4(6):1972-1980
The effect of SDS, pD, and temperature on the structure and stability of the protein disulfide oxidoreductase from Pyrococcus furiosus (PfPDO) was investigated by molecular dynamic (MD) simulations and FT-IR spectroscopy. pD affects the thermostability of alpha-helices and beta-sheets differently, and 0.5% or higher SDS concentration influences the structure significantly. The experiments allowed us to detect a secondary structural reorganization at a definite temperature and pD which may correlate with a high ATPase activity of the protein. The MD simulations supported the infrared data and revealed the different behavior of the N and C terminal segments, as well as of the two active sites. 相似文献
53.
Ilari A Latella MC Ceci P Ribacchi F Su M Giangiacomo L Stefanini S Chasteen ND Chiancone E 《Biochemistry》2005,44(15):5579-5587
The role of the ferroxidase center in iron uptake and hydrogen peroxide detoxification was investigated in Listeria innocua Dps by substituting the iron ligands His31, His43, and Asp58 with glycine or alanine residues either individually or in combination. The X-ray crystal structures of the variants reveal only small alterations in the ferroxidase center region compared to the native protein. Quenching of the protein fluorescence was exploited to assess stoichiometry and affinity of metal binding. Substitution of either His31 or His43 decreases Fe(II) affinity significantly with respect to wt L. innocua Dps (K approximately 10(5) vs approximately 10(7) M(-)(1)) but does not alter the binding stoichiometry [12 Fe(II)/dodecamer]. In the H31G-H43G and H31G-H43G-D58A variants, binding of Fe(II) does not take place with measurable affinity. Oxidation of protein-bound Fe(II) increases the binding stoichiometry to 24 Fe(III)/dodecamer. However, the extent of fluorescence quenching upon Fe(III) binding decreases, and the end point near 24 Fe(III)/dodecamer becomes less distinct with increase in the number of mutated residues. In the presence of dioxygen, the mutations have little or no effect on the kinetics of iron uptake and in the formation of micelles inside the protein shell. In contrast, in the presence of hydrogen peroxide, with increase in the number of substitutions the rate of iron oxidation and the capacity to inhibit Fenton chemistry, thereby protecting DNA from oxidative damage, appear increasingly compromised, a further indication of the role of ferroxidation in conferring peroxide tolerance to the bacterium. 相似文献
54.
Sipione S Ewen C Shostak I Michalak M Bleackley RC 《Journal of immunology (Baltimore, Md. : 1950)》2005,174(6):3212-3219
Calreticulin is an endoplasmic reticulum-resident chaperone that is stored in the cytotoxic granules of CTLs and NK cells and is released with granzymes and perforin upon recognition of target cells. To investigate the role of calreticulin in CTL-mediated killing, we generated CTL lines from crt(+/+) and crt(-/-) mice expressing a constitutively active form of calcineurin in the heart. Crt(-/-) CTLs showed reduced cytotoxic activity toward allogeneic target cells despite normal production, intracellular localization, and activity of granzymes and despite perforin overexpression. Comparable or higher amounts of granzymes were degranulated by crt(-/-) cells in response to immobilized anti-CD3 Abs, indicating that calreticulin is dispensable for the signal transduction that leads to granule exocytosis. The ability to form conjugates with target cells was affected in the crt(-/-) CTLs, explaining the observed reduction in cytotoxicity. Conjugate formation and cytotoxicity were completely restored by treatments that facilitate recognition and contact with target cells, a prerequisite for degranulation and killing. Therefore, we conclude that calreticulin is dispensable for the cytolytic activity of granzymes and perforin, but it is required for efficient CTL-target cell interaction and for the formation of the death synapse. 相似文献
55.
Leone M Di Lello P Ohlenschläger O Pedone EM Bartolucci S Rossi M Di Blasio B Pedone C Saviano M Isernia C Fattorusso R 《Biochemistry》2004,43(20):6043-6058
No general strategy for thermostability has been yet established, because the extra stability of thermophiles appears to be the sum of different cumulative stabilizing interactions. In addition, the increase of conformational rigidity observed in many thermophilic proteins, which in some cases disappears when mesophilic and thermophilic proteins are compared at their respective physiological temperatures, suggests that evolutionary adaptation tends to maintain corresponding states with respect to conformational flexibility. In this study, we accomplished a structural analysis of the K18G/R82E Alicyclobacillus acidocaldarius thioredoxin (BacTrx) mutant, which has reduced heat resistance with respect to the thermostable wild-type. Furthermore, we have also achieved a detailed study, carried out at 25, 45, and 65 degrees C, of the backbone dynamics of both the BacTrx and its K18G/R82E mutant. Our findings clearly indicate that the insertion of the two mutations causes a loss of energetically favorable long-range interactions and renders the secondary structure elements of the double mutants more similar to those of the mesophilic Escherichia coli thioredoxin. Moreover, protein dynamics analysis shows that at room temperature the BacTrx, as well as the double mutant, are globally as rigid as the mesophilic thioredoxins; differently, at 65 degrees C, which is in the optimal growth temperature range of A. acidocaldarius, the wild-type retains its rigidity while the double mutant is characterized by a large increase of the amplitude of the internal motions. Finally, our research interestingly shows that fast motions on the pico- to nanosecond time scale are not detrimental to protein stability and provide an entropic stabilization of the native state. This study further confirms that protein thermostability is reached through diverse stabilizing interactions, which have the key role to maintain the structural folding stable and functional at the working temperature. 相似文献
56.
The antibiotic polymyxin B modulates P2X7 receptor function 总被引:3,自引:0,他引:3
Ferrari D Pizzirani C Adinolfi E Forchap S Sitta B Turchet L Falzoni S Minelli M Baricordi R Di Virgilio F 《Journal of immunology (Baltimore, Md. : 1950)》2004,173(7):4652-4660
The natural peptide polymyxin B (PMB) is a well-known and potent antibiotic that binds and neutralizes bacterial endotoxin (LPS), thus preventing its noxious effects among LPS-mediated endotoxin shock in animal models. We have investigated the effect of PMB on responses mediated by the P2X(7)R in HEK293 and K562 cells transfected with P2X(7) cDNA and in mouse and human macrophages. In addition, in view of the potential exploitation of P2X(7)-directed agonists in antitumor therapy, we also investigated the effect of PMB in B lymphocytes from patients affected by chronic lymphocytic leukemia. PMB, at an optimal concentration dependent on the given cell type, greatly potentiated the effect of nucleotide-mediated P2X(7) stimulation. In particular, ATP-mediated Ca(2+) influx, plasma membrane permeabilization, and cytotoxicity were enhanced to an extent that, in the presence of PMB, cells were killed by otherwise ineffective nucleotide concentrations. The synergistic effect due to the combined application of ATP and PMB was prevented by incubation with the irreversible P2X blocker oxidized ATP (oATP), but not with the reversible antagonist 1-(N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl)-4-phenilpiperazine (KN-62). Cells lacking P2X(7) were fully insensitive to the combined stimulation with PMB and ATP. Furthermore, PMB at the concentrations used had no untoward effects on cell viability. These results point to PMB as a useful tool for the modulation of P2X(7)R function and suggest that care should be used in the evaluation of ATP-stimulated immune cell responses in the presence of PMB as they may not solely be affected by removal of contaminating LPS. 相似文献
57.
Disappearance of the budding yeast Bub2-Bfa1 complex from the mother-bound spindle pole contributes to mitotic exit
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Fraschini R D'Ambrosio C Venturetti M Lucchini G Piatti S 《The Journal of cell biology》2006,172(3):335-346
Budding yeast spindle position checkpoint is engaged by misoriented spindles and prevents mitotic exit by inhibiting the G protein Tem1 through the GTPase-activating protein (GAP) Bub2/Bfa1. Bub2 and Bfa1 are found on both duplicated spindle pole bodies until anaphase onset, when they disappear from the mother-bound spindle pole under unperturbed conditions. In contrast, when spindles are misoriented they remain symmetrically localized at both SPBs. Thus, symmetric localization of Bub2/Bfa1 might lead to inhibition of Tem1, which is also present at SPBs. Consistent with this hypothesis, we show that a Bub2 version symmetrically localized on both SPBs throughout the cell cycle prevents mitotic exit in mutant backgrounds that partially impair it. This effect is Bfa1 dependent and can be suppressed by high Tem1 levels. Bub2 removal from the mother-bound SPB requires its GAP activity, which in contrast appears to be dispensable for Tem1 inhibition. Moreover, it correlates with the passage of one spindle pole through the bud neck because it needs septin ring formation and bud neck kinases. 相似文献
58.
Identification of a novel human granzyme B inhibitor secreted by cultured sertoli cells 总被引:3,自引:0,他引:3
Sipione S Simmen KC Lord SJ Motyka B Ewen C Shostak I Rayat GR Dufour JM Korbutt GS Rajotte RV Bleackley RC 《Journal of immunology (Baltimore, Md. : 1950)》2006,177(8):5051-5058
Sertoli cells have long since been recognized for their ability to suppress the immune system and protect themselves as well as other cell types from harmful immune reaction. However, the exact mechanism or product produced by Sertoli cells that affords this immunoprotection has never been fully elucidated. We examined the effect of mouse Sertoli cell-conditioned medium on human granzyme B-mediated killing and found that there was an inhibitory effect. We subsequently found that a factor secreted by Sertoli cells inhibited killing through the inhibition of granzyme B enzymatic activity. SDS-PAGE analysis revealed that this factor formed an SDS-insoluble complex with granzyme B. Immunoprecipitation and mass spectroscopic analysis of the complex identified a proteinase inhibitor, serpina3n, as a novel inhibitor of human granzyme B. We cloned serpina3n cDNA, expressed it in Jurkat cells, and confirmed its inhibitory action on granzyme B activity. Our studies have led to the discovery of a new inhibitor of granzyme B and have uncovered a new mechanism used by Sertoli cells for immunoprotection. 相似文献
59.
Background
Pulmonary embolism (PE) is a common and potentially fatal disease that is still underdiagnosed. The objective of our study was to reappraise the clinical presentation of PE with emphasis on the identification of the symptoms and signs that prompt the patients to seek medical attention.Methodology/Principal Findings
We studied 800 patients with PE from two different clinical settings: 440 were recruited in Pisa (Italy) as part of the Prospective Investigative Study of Acute Pulmonary Embolism Diagnosis (PISAPED); 360 were diagnosed with and treated for PE in seven hospitals of central Tuscany, and evaluated at the Atherothrombotic Disorders Unit, Firenze (Italy), shortly after hospital discharge. We interviewed the patients directly using a standardized, self-administered questionnaire originally utilized in the PISAPED. The two samples differed significantly as regards age, proportion of outpatients, prevalence of unprovoked PE, and of active cancer. Sudden onset dyspnea was the most frequent symptom in both samples (81 and 78%), followed by chest pain (56 and 39%), fainting or syncope (26 and 22%), and hemoptysis (7 and 5%). At least one of the above symptoms was reported by 756 (94%) of 800 patients. Isolated symptoms and signs of deep vein thrombosis occurred in 3% of the cases. Only 7 (1%) of 800 patients had no symptoms before PE was diagnosed.Conclusions/Significance
Most patients with PE feature at least one of four symptoms which, in decreasing order of frequency, are sudden onset dyspnea, chest pain, fainting (or syncope), and hemoptysis. The occurrence of such symptoms, if not explained otherwise, should alert the clinicians to consider PE in differential diagnosis, and order the appropriate objective test. 相似文献60.
Benicchi T Iozzi S Svahn A Axelsson H Mori E Bernocco S Cappelli F Caramelli C Fanti P Genesio E Maccari L Markova N Micco I Porcari V Schultz J Fecke W 《Journal of biomolecular screening》2012,17(7):933-945
The TWEAK-Fn14 pathway is upregulated in models of inflammation, autoimmune diseases, and cancer. Both TWEAK and Fn14 show increased expression also in the CNS in response to different stimuli, particularly astrocytes, microglia, and neurons, leading to activation of NF-κB and release of proinflammatory cytokines. Although neutralizing antibodies against these proteins have been shown to have therapeutic efficacy in animal models of inflammation, no small-molecule therapeutics are yet available. Here, we describe the development of a novel homogeneous time-resolved fluorescence (HTRF)-based screening assay together with several counterassays for the identification of small-molecule inhibitors of this protein-protein interaction. Recombinant HIS-TWEAK and Fn14-Fc proteins as well as FLAG-TWEAK and Fn14-FLAG proteins and an anti-Fn14 antibody were used to establish and validate these assays and to screen a library of 60 000 compounds. Two HTRF counterassays with unrelated proteins in the same assay format, an antiaggregation assay and a redox assay, were applied to filter out potential false-positive compounds. The novel assay and associated screening cascade should be useful for the discovery of small-molecule inhibitors of the TWEAK-Fn14 protein interaction. 相似文献