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81.
Construction and characterization of affibody-Fc chimeras produced in Escherichia coli 总被引:1,自引:0,他引:1
Rönnmark J Hansson M Nguyen T Uhlén M Robert A Ståhl S Nygren PA 《Journal of immunological methods》2002,261(1-2):199-211
Affibody-Fc chimeras were constructed by genetic fusion between different affibody affinity proteins with prescribed specificities and an Fc fragment derived from human IgG. Using affibody ligands previously selected for binding to respiratory syncytial virus (RSV) surface protein G and Thermus aquaticus (Taq) DNA polymerase, respectively, affibody-Fc fusion proteins showing spontaneous Fc fragment-mediated homodimerization via disulfide bridges were produced in Escherichia coli and affinity purified on protein A Sepharose from bacterial periplasms at yields ranging between 1 and 6 mg/l culture. Further characterization of the chimeras using biosensor technology showed that the affibody moieties have retained high selectivities for their respective targets after fusion to the Fc fragment. Avidity effects in the target binding were observed for the affibody-Fc chimeras compared to monovalent affibody fusion proteins, indicating that both affibody moieties in the chimeras were accessible and contributed in the binding. Fusion of a head-to-tail dimeric affibody moiety to the Fc fragment resulted in tetravalent affibody constructs which showed even more pronounced avidity effects. In addition, the Fc moiety of the chimeras was demonstrated to be specifically recognized by anti-human IgG antibody enzyme conjugates. One application for this class of "artificial antibodies" was demonstrated in a western blotting experiment in which one of the anti-RSV surface protein G affibody-Fc chimeras was demonstrated to be useful for specific detection of the target protein in a complex background consisting of a total E. coli lysate. The results show that through the replacement of the Fab portion of an antibody for an alternative binding domain based on a less complicated structure, chimeric proteins compatible with bacterial production routes containing both antigen recognition domains and Fc domains can be constructed. Such "artificial antibodies" should be interesting alternatives to, for example, whole antibodies or scFv-Fc fusions as detection devices and in diagnostic or therapeutic applications. 相似文献
82.
sa Andersson Susanne M. Grunewald Albert Duschl Alain Fischer James P. Disanto 《European journal of immunology》1997,27(7):1762-1768
The common γ chain (γc) forms a critical component of the receptors for interleukins (IL)-2, IL-4, IL-7, IL-9, and IL-15. We analyzed γc-deficient mice to define a role for γc signaling in the development and function of the macrophage lineage. No major differences in absolute cell numbers, cell surface phenotype, or in vitro function of γ?c compared to γ+c macrophages were observed. We therefore conclude that signaling through the γc chain is not essential for the differentiation of mouse macrophages. Although B and T cells require γc for IL-4 responses, IL-4 up-regulated major histocompatibility class II molecules and inhibited nitric oxide production from γ?c macrophages following stimulation with lipopolysaccharide and interferon-γ. γ?c macrophages could also respond to IL-13, consistent with the model of a type II IL-4 receptor α/IL-13R which can function in the absence of γc. Both IL-4 and IL-13 responses could be completely inhibited with the mouse IL-4 antagonist QY, suggesting that all of the observed IL-13 responses pass through the type II receptor, making it the primary signaling receptor complex for IL-13 in mouse macrophages. 相似文献
83.
Filliol I Sola C Legrand E Rastogi N 《Bulletin de la Societe de pathologie exotique (1990)》1999,92(5):292-298
This investigation dealt with 226 strains (1 isolate/patient) of Mycobacterium tuberculosis isolated in the French West Indies and French Guiana over a three-year period (1994-1996). The genotypic diversity of the isolates was investigated using various molecular markers; essentially two PCR-based rapid methods, namely spoligotyping and double-repetitive-element (DRE)-PCR, as well as three restriction fragment length polymorphism (RFLP)-based methods, namely IS6110-RFLP, DR-RFLP and PGRS-RFLP. Out of 226 isolates investigated, a total of 166 isolates were distributed in 31 spoligotype-defined clusters containing 2-31 strains, which corresponded to a rate of 73% of primary clustering. After secondary typing with DRE-PCR, IS6110-RFLP, DR-RFLP and/or PGRS-RFLP, molecular clonality was established for 73 isolates organised in 25 clusters (32% of clustered isolates). Considering one reactivation case per cluster, the rate of recent transmission was estimated to a minimal rate of 21%, however the available epidemiologic information led to the positive conclusion for only 14% of cases. The data obtained demonstrated the presence of common genotypes of M. tuberculosis among the three overseas French territories, i.e. Guadeloupe, Martinique and French Guiana. The results obtained during this retrospective study clearly indicate the importance of future prospective epidemiological investigations around the clustered cases of tuberculosis, so as to detect the persisting foci of endemic disease and characterize the chain of transmission as well as the subpopulations which are at an increased risk of contracting and/or propagating the disease. Last but not least, the present study also deals with a first phylogenetic approach of M. tuberculosis based on a comparison of the spoligotyping results obtained locally with those reported elsewhere in the world. 相似文献
84.
Paolo Sportoletti Beatrice Del Papa Mariangela De Ioanni Lorenzo Moretti Elisabetta Bonifacio Vania Lanterna Alain Bell Katia Fettucciari Eugenia Carnevali Tiziana Zei Franca Falzetti Massimo F Martelli Antonio Tabilio Mauro Di Ianni 《Biology of blood and marrow transplantation》2006,12(12):1250-1260
T-cell homeostasis is regulated by several molecules; among these, interleukin (IL)-7 plays an essential role in the survival and homeostatic proliferation of peripheral naive T cells. In a previous study, we investigated whether human mesenchymal stromal cells (MSCs) could be engineered with the IL-7 gene to produce functional level of this cytokine. In the present study, we analyzed the impact of different quantities of IL-7 produced by MSCs on the survival and proliferation of a negative immunoselected naive (CD3(+)/CD45RA(+)) T-cell population. Co-cultivation of peripheral naive T cells with MSCs producing low (16 pg/mL) or high (1000 pg/mL) IL-7 levels or in the presence of exogenous IL-7 (0.01 ng/mL and 100 ng/mL) maintained the CD3(+)/CD45RA(+) naive T-cell phenotype. Chemokine receptor CCR7(+) expression was also maintained among this T-cell population. Naive T-cell molecular characteristics were maintained as assessed by the Vbeta spectratyping complexity score, which showed the maintenance of a broad T-cell repertoire. No Th1 or Th2 differentiation was observed, as assessed by interferon-gamma or IL-4 accumulation. In contrast, only MSCs producing high amounts of IL-7 caused increased activation (CD25 31.2% +/- 12% vs 10% +/- 3.5%; P < .05), proliferation (CD71 17.8+/-7% vs 9.3%+/-3, P < .05), apoptosis (assessed by annexin V: 18.6% +/- 5% vs 14.9% +/- 2.6%; P > .05), and the phase S cell cycle (15% vs 6.9%, P > .05). Exogenous IL-7 exhibited no significant effect. In conclusion, we demonstrated that IL-7 produced by MSCs has a dose-independent effect on naive T-cell survival while exerting a dose-dependent effect on activation/proliferation. Due to the continuous production of IL-7 by engineered cells, our system is more efficacious than exogenous IL-7. 相似文献
85.
Jean -Paul Richalet Claude Soulard Alain Nitenberg Bernard Teisseire Jean de Bovée Sadino Séroussi 《European journal of applied physiology》1981,47(1):27-39
Summary Conditions of oxygen extraction by the myocardium have been studied in 12 subjects (44±9 years old) with pure mitral stenosis without clinical, metabolic or electrical sign of coronary insufficiency.Oxygen-hemoglobin equilibrium curves (OHEC) have been determined on arterial, mixed venous and coronary sinus blood, at rest and during a moderate (60 W, 10 min) exercise performed on a bicycle ergometer in supine position. Physiological values, at rest and during exercise, of the following functional parameters of the OHEC were determined in the in-vivo conditions of pH and PCO2P50, nHill, DS max = maximal value of OHEC slope (DS=SO2/PO2), PDS max, SDS max. The concentration of plasma electrolytes capable to modify one of these parameters was controlled in each blood sample.In coronary sinus blood, P50 rises from 27.5±1.7 to 28.9±1.6 Torr during exercise (p<0.01). At rest, Hill's n in myocardial venous blood (2.67±0.09) is significantly higher than in arterial blood (2.61±0.08, p<0.01). A decrease in DS max (2.67±0.20 to 2.53±0.12%. Torr–1; p<0.01) and an increase in PDS max (20.9±1.9 to 22.6±2.1 Torr, p<0.01) are observed. At rest, the myocardial venous point (PcsO2, ScsO2) is not significantly distinct from the maximal slope point (PDS max, SDS max). After 1 min of exercise, a small gap appears which becomes significant at the end of the exercise (PDS max-PcsO2=0.2 Torr at rest, 2.7 Torr after exercise; SDS max-ScsO2=4.1% at rest, 7.9% after exercise). DS value at the myocardial venous point is only 2% at rest and 6% after exercise, lower than its maximal value.The gap between venous point and DS max point could constitute an error signal in the regulation loop of the coronary circulation. The existence of a physiological receptor, sensitive to instant variations in myocardial tissue PO2 and able to maintain venous point near DS max point could be considered. 相似文献
86.
87.
Mehnert P Malchaire J Kampmann B Piette A Griefahn B Gebhardt H 《European journal of applied physiology》2000,82(1-2):52-60
The prediction of the mean skin temperature used for the Required Sweat Rate index was criticised for not being valid in
conditions with high radiation and high humidity. Based on a large database provided by 9 institutes, 1999 data points obtained
using steady-state conditions, from 1399 experiments and involving 377 male subjects, were used for the development of a new
prediction model. The observed mean skin temperatures ranged from 30.7 °C to 38.6 °C. Experimental conditions included air
temperatures (T
a) between 20 and 55 °C, mean radiant temperatures (T
r) up to 145 °C, partial vapour pressures (P
a) from 0.2 to 5.3 kPa, air velocities (v
a) between 0.1 and 2 m/s, and metabolic rates (M) from 102 to 620 W. Rectal temperature (T
re) was included in the models to increase the accuracy of prediction. Separate models were derived for nude (clothing insulation,
Icl, ≤0.2 clo, where 1 clo=0.155 m2 · °C · W−1, which is equivalent to the thermal insulation of clothing necessary to maintain a resting subject in comfort in a normally
ventilated room, air movement=10 cm/s, at a temperature of 21 °C and a humidity of less than 50%) and clothed (0.6 ≤ Icl ≤ 1.0 clo) subjects using a multiple linear regression technique with re-sampling (non-parametric bootstrap). The following
expressions were obtained for nude and clothed subjects, respectively: T
sk=7.19 + 0.064T
a + 0.061T
r + 0.198P
a− 0.348v
a + 0.616T
re and T
sk=12.17 + 0.020T
a + 0.044T
r + 0.194P
a − 0.253v
a + 0.0029M + 0.513T
re. For the nude and clothed subjects, 83.3% and 81.8%, respectively, of the predicted skin temperatures were within the range
of ±1 °C of the observed skin temperatures. It is concluded that the proposed models for the prediction of the mean skin temperature
are valid for a wide range of warm and hot ambient conditions in steady-state conditions, including those of high radiation
and high humidity.
Accepted: 7 February 2000 相似文献
88.
Vergu E Mallet A Golmard JL 《Journal of acquired immune deficiency syndromes (1999)》2002,30(3):263-270
OBJECTIVE: To study the role of resistance characteristics of viral mutants in the prediction of virologic and immunologic response to antiretroviral therapy in HIV-infection. METHODS: This study is based on a mathematical model that generates viral and immunologic dynamics of HIV infection, taking into account drug-resistant mutants and therapy. We analyzed predictive factors of the increase in CD4 cell count and of the decrease in viral load from baseline after 6 months of HAART on a sample of 300 simulated individuals. The set of potential predictors was constituted by patients' state at initiation of therapy and by resistance characteristics of viral strains at that time. Predictive models, obtained by stepwise regression, were selected and compared using Mallows' Cp criterion. RESULTS: In addition to baseline viral load and CD4 cell count, known to influence response to therapy, baseline CD8 cell count and resistance characteristics of detectable strains are shown to improve the accuracy of the prediction. On the contrary, resistance parameters of low frequency viral mutants have no predictive value. CONCLUSIONS: Characteristics of preexisting detectable resistant mutants as determinants of virologic and immunologic response to antiretroviral therapy increase the capacity to predict the outcome of the treatment. Therefore, the use of phenotypic and genotypic testing could be crucial and should be considered for the choice of therapy. 相似文献
89.
Lura Brianna Caddle Jeremy L. Grant Jin Szatkiewicz Johann van Hase Bobbi-Jo Shirley Joerg Bewersdorf Christoph Cremer Alain Arneodo Andre Khalil Kevin D. Mills 《Chromosome research》2007,15(8):1061-1073
Radiation exposure is an occupational hazard for military personnel, some health care professionals, airport security screeners,
and medical patients, with some individuals at risk for acute, high-dose exposures. Therefore, the biological effects of radiation,
especially the potential for chromosome damage, are major occupational and health concerns. However, the biophysical mechanisms
of chromosome instability subsequent to radiation-induced DNA damage are poorly understood. It is clear that interphase chromosomes
occupy discrete structural and functional subnuclear domains, termed chromosome territories (CT), which may be organized into
‘neighborhoods’ comprising groups of specific CTs. We directly evaluated the relationship between chromosome positioning,
neighborhood composition, and translocation partner choice in primary lymphocytes, using a cell-based system in which we could
induce multiple, concentrated DNA breaks via high-dose irradiation. We critically evaluated mis-rejoining profiles and tested
whether breaks occurring nearby were more likely to fuse than breaks occurring at a distance. We show that CT neighborhoods
comprise heterologous chromosomes, within which inter-CT distances directly relate to translocation partner choice. These
findings demonstrate that interphase chromosome arrangement is a principal factor in genomic instability outcomes in primary
lymphocytes, providing a structural context for understanding the biological effects of radiation exposure, and the molecular
etiology of tumor-specific translocation patterns. 相似文献
90.
Control of interneurone firing pattern by axonal autoreceptors in the juvenile rat cerebellum 总被引:2,自引:0,他引:2
Recent work has shown that certain neurones have axonal GABAA receptors, whose tonic activation modifies their firing properties and neurotransmitter release capability. In addition, results obtained in interneurones of the molecular layer of the cerebellum indicate that action potential-released GABA binds back to the axon that released it, generating an autoreceptor current. In the present paper, we show that at physiological Cl− i concentration (15 m m ) and at 34–36°C, the autoreceptor current generates a large amplitude (up to 21 mV) afterdepolarization that lasts for about 150 ms, and that occasionally leads to double firing. Furthermore we show that elimination of the afterdepolarization, by either blocking GABAA receptors, or eliminating the autoreceptor currents through prolonged whole-cell recording, decreases burst firing. I h (a hyperpolarization-activated current) was previously found to be prominent in interneurone axons. We show that blocking I h leads to an increase in the amplitude of the autoreceptor current as well as of the associated afterdepolarization, suggesting a shunting effect of I h on autoreceptor-mediated afterdepolarization. Conversely, blocking I h accentuates burst firing. The effects of autoreceptor-mediated afterdepolarization on firing are prominent during a period of development when interneurone synapses are stabilized and vanish by postnatal day 17 (PN 17), together with the expression of the autoreceptor current. Altogether, this work reveals a new role for autoreceptors in the regulation of cell excitability and firing pattern, which may contribute to the development and stabilization of the cerebellar network. 相似文献