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51.
Haddad P Noël J Vadnais J Mathé L Vallerand D 《Canadian journal of physiology and pharmacology》1999,77(11):852-859
Liver cell pH and volume regulation are perturbed by prolonged cold storage in University of Wisconsin solution and subsequent rewarming, but the molecular basis of this effect remains unknown. We prepared membranes from hepatocytes subjected to variable periods of cold preservation with or without subsequent rewarming and probed them by Western blotting with specific antibodies against the Na+ -H+ exchanger isoform NHE-1 and the Na+ -K+ ATPase alpha subunit. Results were compared with the content of GLUT-2, an abundant basolateral protein. NHE-1 decreased significantly as cold preservation times exceeded 10 h. Subsequent rewarming by short-term culture at 37 degrees C did not further reduce this parameter. On the other hand, expression of Na+ -K+ ATPase remained stable during cold storage times lasting up to 48 h, whereas rewarming resulted in a dramatic reduction in cells cold preserved beyond 10 h. In contrast, the membrane content of GLUT-2 was unaffected by cold preservation with or without subsequent rewarming. The results indicate that cold storage and rewarming respectively and selectively modulate the expression of specific hepatocellular membrane transport proteins. 相似文献
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53.
Hallez S Simon P Maudoux F Doyen J Noël JC Beliard A Capelle X Buxant F Fayt I Lagrost AC Hubert P Gerday C Burny A Boniver J Foidart JM Delvenne P Jacobs N 《Cancer immunology, immunotherapy : CII》2004,53(7):642-650
Purpose: Infection with oncogenic human papillomavirus (HPV) and HPV-16 in particular is a leading cause of anogenital neoplasia. High-grade intraepithelial lesions require treatment because of their potential to progress to invasive cancer. Numerous preclinical studies have demonstrated the therapeutic potential of E7-directed vaccination strategies in mice tumour models. In the present study, we tested the immunogenicity of a fusion protein (PD-E7) comprising a mutated HPV-16 E7 linked to the first 108 amino acids of Haemophilus influenzae protein D, formulated in the GlaxoSmithKline Biologicals adjuvant AS02B, in patients bearing oncogenic HPV-positive cervical intraepithelial neoplasia (CIN). Methods: Seven patients, five with a CIN3 and two with a CIN1, received three intramuscular injections of adjuvanted PD-E7 at 2-week intervals. Three additional CIN1 patients received a placebo. CIN3 patients underwent conization 8 weeks postvaccination. Cytokine flow cytometry and ELISA were used to monitor antigen-specific cellular and antibody responses from blood taken before and after vaccine or placebo injection. Results: Some patients had preexisting systemic IFN- CD4+ (1/10) and CD8+ (5/10) responses to PD-E7. Vaccination, not placebo injection, elicited systemic specific immune responses in the majority of the patients. Five vaccinated patients (71%) showed significantly increased IFN- CD8+ cell responses upon PD-E7 stimulation. Two responding patients generated long-term T-cell immunity toward the vaccine antigen and E7 as well as a weak H. influenzae protein D (PD)–directed CD4+ response. All the vaccinated patients, but not the placebo, made significant E7- and PD-specific IgG. Conclusions: The encouraging results obtained from this study performed on a limited number of subjects justify further analysis of the efficacy of the PD-E7/AS02B vaccine in CIN patients. 相似文献
54.
55.
Noë A Verhofstede C Plum J 《Journal of biological regulators and homeostatic agents》2004,18(2):178-182
In the last two decades we have witnessed the progression of a newly introduced infection in humans. It is sobering that despite a world-wide effort and the tremendous progression of technical capabilities and scientific knowledge we are still not able to control the global epidemic of HIV. In 2004 more than 40 million people were infected. Educational approaches to modify risk-taking behavior is still the most critical component of prevention and the most important measure to limit the spread of the infection. Vaccine development, which is still far from promising, is probably the only way to control the disease in the future. 相似文献
56.
Davezac N Tondelier D Lipecka J Fanen P Demaugre F Debski J Dadlez M Schrattenholz A Cahill MA Edelman A 《Proteomics》2004,4(12):3833-3844
Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF gene (cftr). Physiologically, CF is characterized by an abnormal chloride secretion in epithelia due to a dysfunction of a mutated cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a cAMP-dependent chloride channel whose most frequent mutation, deltaF508, leads to an aberrantly folded protein which causes a dysfunction of the channel. However, a growing number of reports suggest that modifier genes and environmental factors are involved in the physiology of CF. To identify proteins whose expression depends on wild-type WT-CFTR or deltaF508-CFTR, we chose a global proteomic approach based on the use of two-dimensional gel electrophoresis (2-DE) and mass spectrometry. The experiments were carried out with HeLa cells stably transfected with WT-CFTR (pTCFWT) or deltaF508-CFTR (pTCFdeltaF508). These experiments unmasked keratin 8 (K8) and 18 (K18) which were differentially expressed in pTCFWT vs. pTCFdeltaF508. An immunoblot of K18 confirmed the 2-DE results. Intracellular localization experiments of WT-CFTR, deltaF508-CFTR, K8, and K18 suggest that the expression of these proteins are linked, and that the concentrations of K8 and K18 and/or their distribution may be involved in the traffic of WT-CFTR/deltaF508-CFTR. A functional assay for CFTR revealed that specifically lowering K18 expression or changing its distribution leads to the delivery of functional deltaF508-CFTR to the plasma membrane. This work suggests a novel function of K18 in CF. 相似文献
57.
Increased zinc content in transplastomic tobacco plants expressing a polyhistidine-tagged Rubisco large subunit 总被引:2,自引:0,他引:2
Rumeau D Bécuwe-Linka N Beyly A Carrier P Cuiné S Genty B Medgyesy P Horvath E Peltier G 《Plant biotechnology journal》2004,2(5):389-399
Rubisco is a hexadecameric enzyme composed of two subunits: a small subunit (SSU) encoded by a nuclear gene (rbcS), and a large subunit (LSU) encoded by a plastid gene (rbcL). Due to its high abundance, Rubisco represents an interesting target to express peptides or small proteins as fusion products at high levels. In an attempt to modify the plant metal content, a polyhistidine sequence was fused to Rubisco, the most abundant protein of plants. Plastid transformation was used to express a polyhistidine (6x) fused to the C-terminal extremity of the tobacco LSU. Transplastomic tobacco plants were generated by cotransformation of polyethylene glycol-treated protoplasts using two vectors: one containing the 16SrDNA marker gene, conferring spectinomycin resistance, and the other the polyhistidine-tagged rbcL gene. Homoplasmic plants containing L8-(His)6S8 as a single enzyme species were obtained. These plants contained normal Rubisco amounts and activity and displayed normal photosynthetic properties and growth. Interestingly, transplastomic plants accumulated higher zinc amounts than the wild-type when grown on zinc-enriched media. The highest zinc increase observed exceeded the estimated chelating ability of the polyhistidine sequence, indicating a perturbation in intracellular zinc homeostasis. We discuss the possibility of using Rubisco to express foreign peptides as fusion products and to confer new properties to higher plants. 相似文献
58.
Folschweiller N Pacaud K Celia H Potier N Cobessi D Van Dorsselaer A Pattus F 《Protein expression and purification》2004,38(1):79-83
The number of protein structures solved using multiwavelength anomalous diffraction methods coupled with selenomethionine substitution has grown dramatically over the last years. We show using the outer membrane pyoverdin receptor FpvA that Pseudomonas aeruginosa can be used for producing proteins with a high level of selenomethionine incorporation. To circumvent problems encountered with mass spectroscopy analysis of purified membrane proteins, in-gel trypsin digestion of FpvA coupled with MALDI mass spectrometry analysis of the resulting peptides was used to determine the extent of selenomethionine incorporation. Selenomethionine incorporation greater than 95% was achieved using P. aeruginosa as an overexpression system. 相似文献
59.
Mathieu N Spicuglia S Gorbatch S Cabaud O Fernex C Verthuy C Hempel WM Hueber AO Ferrier P 《The Journal of biological chemistry》2003,278(20):18101-18109
To assess the role of the T cell receptor (TCR) beta gene enhancer (Ebeta) in regulating the processing of VDJ recombinase-generated coding ends, we assayed TCRbeta rearrangement of Ebeta-deleted (DeltaEbeta) thymocytes in which cell death is inhibited via expression of a Bcl-2 transgene. Compared with DeltaEbeta, DeltaEbeta Bcl-2 thymocytes show a small accumulation of TCRbeta standard recombination products, including coding ends, that involves the proximal Dbeta-Jbeta and Vbeta14 loci but not the distal 5' Vbeta genes. These effects are detectable in double negative pro-T cells, predominate in double positive pre-T cells, and correlate with regional changes in chromosomal structure during double negative-to-double positive differentiation. We propose that Ebeta, by driving long range nucleoprotein interactions and the control of locus expression and chromatin structure, indirectly contributes to the stabilization of coding ends within the recombination processing complexes. The results also illustrate Ebeta-dependent and -independent changes in chromosomal structure, suggesting distinct modes of regulation of TCRbeta allelic exclusion depending on the position within the locus. 相似文献
60.
Hwang KJ Xiang B Gruschus JM Nam KY No KT Nirenberg M Ferretti JA 《Biochemistry》2003,42(43):12522-12531
The three-dimensional solution structure obtained by NMR of the A35T mutant vnd/NK-2 homeodomain bound to the vnd/NK-2 consensus 16 bp DNA sequence was determined. This mutation to threonine from alanine in position 35 in helix II of the vnd/NK-2 homeodomain is associated with early embryonic lethality in Drosophila melanogaster. Although the unbound mutant protein is not structured, in the DNA-bound state it adopts the three-helix fold characteristic of all known homeodomains, but with alterations relative to the structure of the wild-type analogue. These structural modifications occur, and are accompanied by a 50-fold reduction in the DNA binding affinity, even though most of the protein-DNA interactions originally seen for the wild-type homeodomain are found likewise in the threonine analogue. Alterations include torsional angle changes in the loop between helix I and helix II, and in the turn between helix II and helix III, as well as in a distortion of the usual antiparallel orientation of helix I with respect to helix II. The alteration of the position of leucine 40 in the A35T mutant is proposed to explain the observed 1.27 ppm upfield shift of the corresponding amide proton resonance relative to the value observed for the wild-type analogue. A detailed comparison of the structures of the mutant A35T and wild-type vnd/NK-2 homeodomains bound to the cognate DNA is presented. The consequences of the structural alteration of the DNA-bound A35T mutant vnd/NK-2 protein may constitute the basis of the observed early embryonic lethality. 相似文献