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121.
Abad JL Serrano F San Román AL Delgado R Bernad A González MA 《The journal of gene medicine》2002,4(1):27-37
Background
Retroviral transduction of human peripheral blood T cells has considerable potential in the development of gene therapy strategies for immunological disorders. New vectors and experimental procedures have been developed for efficient transduction of several genes into human T cells.Methods
Bicistronic retroviral vectors encoding distinct cell markers were used for the simultaneous multiple transduction of a human T‐cell line (MT‐2), as well as of human peripheral blood T cells from normal donors. Transduction efficiencies were evaluated by flow cytometry and double‐ and triple‐transduced cells were isolated by fluorescence cell sorting.Results
Four new bicistronic retroviral vectors were developed that express different gene markers under the control of the internal ribosome entry site (IRES) of the encephalomyocarditis virus. These markers are, respectively, enhanced green fluorescent protein (EGFP), β‐galactosidase, and truncated versions of human nerve growth factor receptor (ΔNGFR) and human growth hormone receptor (ΔGHR). A single 1 h spinoculation infection, performed in the presence of polybrene and using transiently produced amphotropic retroviral particles, was sufficient to obtain transduction efficiencies consistently greater than 50% on human peripheral blood T lymphocytes which had been previously stimulated for 3 days with immobilized anti‐CD3. The transient production of viral particles encoding EGFP, ΔNGFR, and ΔGHR markers in the same viral supernatant has allowed up to three different genes to be introduced simultaneously into human T cells.Conclusions
This study describes new experimental conditions for efficient single‐step multiple transduction of human primary T lymphocytes. The procedure could be of interest for the development of gene therapy approaches. Copyright © 2002 John Wiley & Sons, Ltd.122.
Zavala F Abad S Ezine S Taupin V Masson A Bach JF 《Journal of immunology (Baltimore, Md. : 1950)》2002,168(4):2011-2019
Converging evidence that G-CSF, the hemopoietic growth factor of the myeloid lineage, also exerts anti-inflammatory and pro-Th2 effects, prompted us to evaluate its direct therapeutic potential in autoimmune diseases. Here we report a novel activity of G-CSF in experimental allergic encephalomyelitis, a murine model for multiple sclerosis, driven by Th1-oriented autoaggressive cells. A short 7-day treatment with G-CSF, initiated at the onset of clinical signs, provided durable protection from experimental autoimmune encephalomyelitis. G-CSF-treated mice displayed limited demyelination, reduced recruitment of T cells to the CNS, and very discrete autoimmune inflammation, as well as barely detectable CNS mRNA levels of cytokines and chemokines. In the periphery, G-CSF treatment triggered an imbalance in the production by macrophages as well as autoreactive splenocytes of macrophage inflammatory protein-1alpha and monocyte chemoattractant protein-1, the prototypical pro-Th1 and pro-Th2 CC chemokines, respectively. This chemokine imbalance was associated with an immune deviation of the autoreactive response, with reduced IFN-gamma and increased IL-4 and TGF-beta1 levels. Moreover, G-CSF limited the production of TNF-alpha, a cytokine also associated with early CNS infiltration and neurological deficit. These findings support the potential application of G-CSF in the treatment of human autoimmune diseases such as multiple sclerosis, taking advantage of the wide clinical favorable experience with this molecule. 相似文献
123.
Belkhodja Ramzi Morales Fermín Quílez Rebeca López-Millán Ana Flor Abadía Anunciación Abadía Javier 《Photosynthesis research》1998,56(3):265-276
Iron deficiency was found to affect the redox state of the Photosystem II acceptor side in dark-adapted, attached leaves of sugar beet (Beta vulgaris L.). Dark-adapted iron-deficient leaves exhibited relatively high Fo and Fpl levels in the Kautsky chlorophyll fluorescence induction curve when compared to the iron-sufficient controls. However, far-red illumination led to marked decreases in the apparent Fo and Fpl levels. Modulated fluorescence showed that far-red light decreased the fluorescence yield to the true Fo levels by increasing photochemical quenching, without inducing changes in the level of non-photochemical quenching. In dark-adapted, iron-deficient leaves, far-red illumination induced a faster fluorescence decay in the µs-ms time domain, indicating an improvement in the electron transport after the primary quinone acceptor in the reducing side of Photosystem II. All these data indicate that in iron-deficient leaves the plastoquinone pool was reduced in the dark. The extent of the plastoquinone reduction in sugar beet depended on the chlorophyll concentration of the leaf, on the time of preillumination and on the duration of dark adaptation. The dark reduction of plastoquinone was observed not only in sugar beet but also in other plant species affected by iron deficiency both in controlled conditions and in the field. 相似文献
124.
The survival of human enteric viruses on several porous (paper and cotton cloth) and nonporous (aluminum, china, glazed tile, latex, and polystyrene) environmental surfaces has been evaluated. Viruses persisted for extended periods on several types of materials commonly found in institutions and domestic environments. The stability of the viruses was generally influenced by environmental factors such as relative humidity (RH), temperature, and the type of surface contaminated. Overall, hepatitis A virus (HAV) and human rotavirus (HRV) were more resistant to inactivation than enteric adenovirus (ADV) and poliovirus (PV). The resistance to the desiccation step appears to be of major significance in determining the survival of a virus dried on fomites. ADV and PV showed a pronounced decrease in titer at this stage, whereas HAV and HRV displayed little decay at the desiccation step. HAV and HRV persistence was not affected by the presence of fecal material. On nonporous surfaces, PV and ADV persisted better in the presence of feces. However, on porous fomites the presence of fecal material had a negative influence on the survival of PV and ADV. Except for HRV, greater virus survival was observed at 4 degrees than at 20 degrees C. PV and HAV survival was enhanced at high RH; the survival of the latter was enhanced at least for nonporous materials. When dried on porous materials, HRV also exhibited greater persistence at high RH. The survival of ADV was not affected by RH. The validity of using bacteriophages of Bacteroides fragilis as indicators of human viruses dried on fomites was evaluated.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
125.
Genomic organization of the halophilic archaeon Haloferax mediterranei: physical map of the chromosome. 总被引:6,自引:0,他引:6
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Pulsed field gel electrophoresis (PFG) has been used to study the genomic organization of the halophilic archaeon Haloferax mediterranei. Analysis of the different genomic elements as well as the restriction patterns obtained with several endonucleases revealed that this microorganism has a circular chromosome of 2.9 Mb and, at least, three extrachromosomal elements of 490, 320 and 130 kb respectively. The complete physical map of the chromosome for the endonucleases PacI and BamHI has been constructed, and several BcII, BgIII and DraI restriction fragments have been aligned on these maps. The localization of heterologous and homologous genes on the physical map, including those for rRNA, lay the ground work for the construction of a genetic map. 相似文献
126.
Characterization of Palo Podrido, a Natural Process of Delignification in Wood 总被引:4,自引:0,他引:4
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Eduardo Agosin Robert A. Blanchette Herman Silva Catherine Lapierre Kory R. Cease Rebecca E. Ibach Andr R. Abad Pedro Muga 《Applied microbiology》1990,56(1):65-74
Chemical and morphological changes of incipient to advanced stages of palo podrido, an extensively delignified wood, and other types of white rot decay found in the temperate forests of southern Chile were investigated. Palo podrido is a general term for white rot decay that is either selective or nonselective for the removal of lignin, whereas palo blanco describes the white decayed wood that has advanced stages of delignification. Selective delignification occurs mainly in trunks of Eucryphia cordifolia and Nothofagus dombeyi, which have the lowest lignin content and whose lignins have the largest amount of β-aryl ether bonds and the highest syringyl/guaiacyl ratio of all the native woods included in this study. A Ganoderma species was the main white rot fungus associated with the decay. The structural changes in lignin during the white rot degradation were examined by thioacidolysis, which revealed that the β-aryl ether-linked syringyl units were more specifically degraded than the guaiacyl ones, particularly in the case of selective delignification. Ultrastructural studies showed that the delignification process was diffuse throughout the cell wall. Lignin was first removed from the secondary wall nearest the lumen and then throughout the secondary wall toward the middle lamella. The middle lamella and cell corners were the last areas to be degraded. Black manganese deposits were found in some, but not all, selectively delignified samples. In advanced stages of delignification, almost pure cellulose could be found, although with a reduced degree of polymerization. Cellulolytic enzymes appeared to be responsible for depolymerization. A high brightness and an easy refining capacity were found in an unbleached pulp made from selectively delignified N. dombeyi wood. Its low viscosity, however, resulted in poor resistance properties of the pulp. The last stage of degradation (i.e., decomposition of cellulose-rich secondary wall layers) resulted in a gelatinlike substance. Ultrastructural and chemical analyses of this substance showed the matrix to have no microfibrillar structure characteristic of woody cell walls but to still be rich in glucan. 相似文献
127.
C. Djian-Caporalino L. Pijarowski A. Januel V. Lefebvre A. Daubèze A. Palloix A. Dalmasso P. Abad 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1999,99(3-4):496-502
Capsicum annuum L. has resistance to root-knot nematodes (RKN) (Meloidogyne spp.), severe polyphagous pests that occur world-wide. Several single dominant genes confer this resistance. Some are highly
specific, whereas others are effective against a wide range of species. The spectrum of resistance to eight clonal RKN populations
of the major Meloidogyne species, M. arenaria (2 populations), M. incognita (2 populations), M. javanica (1 population), and M. hapla (3 populations) was studied using eight lines of Capsicum annuum. Host susceptibility was determined by counting the egg masses (EM) on the roots. Plants were classified into resistant (R;
EM ≤ 5) or susceptible (H; EM >5) classes. The french cultivar Doux Long des Landes was susceptible to all nematodes tested.
The other seven pepper lines were highly resistant to M. arenaria, M. javanica and one population of M. hapla. Variability in resistance was observed for the other two populations of M. hapla. Only lines PM687, PM217, Criollo de Morelos 334 and Yolo NR were resistant to M. incognita. To investigate the genetic basis of resistance in the highly resistant line PM687, the resistance of two progenies was tested
with the two populations of M.
incognita: 118 doubled-haploid (DH) lines obtained by androgenesis from F1 hybrids of the cross between PM687 and the susceptible cultivar Yolo Wonder, and 163 F2 progenies. For both nematodes populations, the segregation patterns 69 R / 49 S for DH lines and 163 R / 45 S for F2 progenies were obtained at 22°C and at high temperatures (32°C and 42°C). The presence of a single dominant gene that totally
prevented multiplication of M. incognita was thus confirmed and its stability at high temperature was demonstrated. This study confirmed the value of C. annuum as a source of complete spectrum resistance to the major RKN.
Received: 2 July 1998 / Accepted: 11 March 1999 相似文献
128.
Abad ML Verdura T Vela A Iglesias MJ Gutiérrez D Veiga M Aguilera A Regueiro BJ 《Molecular biotechnology》2004,28(2):87-95
The pUC-based pNL4-3 plasmid is the most widely used vector for in vitro manipulations of the HIV-1 proviral sequences. We
have developed a minimal plasmid (pCHUS) based on pNL4-3, which may be useful to facilitate the design of HIV-based constructions.
The strategy that has allowed us to construct pCHUS includes the following steps: (1) pNL-3 digestion by using restriction
sites contained within the long terminal repeats (LTRs), (2) recircularization of the fragment containing the pUC18 sequence,
(3) amplification of the LTR region restored in the previous step, (4) double digestion of the products obtained in steps
2 and 3, (5) ligation of the fragment containing ColE1+AmpR with the LTR fragment, (6) linearization of the intermediate plasmid obtained, and (7) insertion of the fragment containing
the proviral genome into the linearized vector. The pCHUS plasmid includes essential information for its replication and antibiotic
selection in bacteria, but it lacks all the unnecessary sequences. Our results suggest that pCHUS may be more advantageous
than pNL4-3 for in vitro manipulation of the HIV-1 proviral genome. In addition, we describe a potential application of this
new vector for pseudotyping HIV-1 particles, using a single plasmid transfection, as a more helpful alternative to the traditionally
used cotransfection method. 相似文献
129.
Lázaro FJ Abadía AR Romero MS Gutiérrez L Lázaro J Morales MP 《Biochimica et biophysica acta》2005,1740(3):434-445
Ex vivo freeze-dried rat muscle tissues, collected at different times t after a single dose of subcutaneously injected iron dextran, have been magnetically characterised. The AC susceptibility of the tissues shows an overall superparamagnetic behaviour and the dependence on t of, especially, the out-of-phase component is remarkably systematic despite the fact that each tissue originates in a different rat individual. The experiments show that the akaganéite (beta-FeOOH) nanoparticles contained in the injected drug are progressively degraded in the living tissue and, at times of the order of 1 month and for all the analysed rat individuals, converge to a magnetically well-defined species with much narrower magnetic activation energy distribution than iron dextran. Thorough transmission electron microscopy experiments of the same tissues indicate the presence of oxyhydroxide particles, whose size decreases for increasing t in agreement with the interpretation of the magnetic susceptibility. The conclusions drawn from the magnetic study do well correspond to the properties of the whole tissue since no biochemical extraction work has been done. The AC susceptibility appears to be a valuable and complementary tool in pharmacological studies of iron-containing drugs. 相似文献
130.
The molecular basis of centromere formation in a particular chromosomal region is not yet understood. In higher eukaryotes,
no specific DNA sequence is required for the assembly of the kinetochore, but similar centromeric chromatins are formed on
different centromere DNA sequences. Although epigenesis has been proposed as the main mechanism for centromere specification,
DNA recognition must also play a role. Through the analysis of Drosophilacentromeric DNA sequences, we found that dodeca satellite and 18HT satellite are able to form unusual DNA structures similar
to those formed by telomeric sequences. These findings suggest the existence of a common centromeric structural DNA motif
which we feel merits further investigation.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献