633 resultados para Reconstitution immunitaire


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Extensive studies of the β-phaseolin (phas) gene in transgenic tobacco have shown that it is highly active during seed embryogenesis but is completely silent in leaf and other vegetative tissues. In vivo footprinting revealed that the lack of even basal transcriptional activity in vegetative tissues is associated with the presence of a nucleosome that is rotationally positioned with base pair precision over three phased TATA boxes present in the phas promoter. Positioning is sequence-dependent because an identical rotational setting is obtained upon nucleosome reconstitution in vitro. A comparison of DNase I and dimethyl sulfate footprints in vivo and in vitro strongly suggests that this repressive chromatin architecture is remodeled concomitant with gene activation in the developing seed. This leads to the disruption of histone-mediated DNA wrapping and the assembly of the TATA boxes into a transcriptionally competent nucleoprotein complex.

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The chromophore of photoactive yellow protein (PYP) (i.e., 4-hydroxycinnamic acid) has been replaced by an analogue with a triple bond, rather than a double bond (by using 4-hydroxyphenylpropiolic acid in the reconstitution, yielding hybrid I) and by a “locked” chromophore (through reconstitution with 7-hydroxycoumarin-3-carboxylic acid, in which a covalent bridge is present across the vinyl bond, resulting in hybrid II). These hybrids absorb maximally at 464 and 443 nm, respectively, which indicates that in both hybrids the deprotonated chromophore does fit into the chromophore-binding pocket. Because the triple bond cannot undergo cis/trans (or E/Z) photoisomerization and because of the presence of the lock across the vinyl double bond in hybrid II, it was predicted that these two hybrids would not be able to photocycle. Surprisingly, both are able. We have demonstrated this ability by making use of transient absorption, low-temperature absorption, and Fourier-transform infrared (FTIR) spectroscopy. Both hybrids, upon photoexcitation, display authentic photocycle signals in terms of a red-shifted intermediate; hybrid I, in addition, goes through a blue-shifted-like intermediate state, with very slow kinetics. We interpret these results as further evidence that rotation of the carbonyl group of the thioester-linked chromophore of PYP, proposed in a previous FTIR study and visualized in recent time-resolved x-ray diffraction experiments, is of critical importance for photoactivation of PYP.

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The human androgen receptor (AR) is a ligand-activated transcription factor that regulates genes important for male sexual differentiation and development. To better understand the role of the receptor as a transcription factor we have studied the mechanism of action of the N-terminal transactivation function. In a protein–protein interaction assay the AR N terminus (amino acids 142–485) selectively bound to the basal transcription factors TFIIF and the TATA-box-binding protein (TBP). Reconstitution of the transactivation activity in vitro revealed that AR142–485 fused to the LexA protein DNA-binding domain was competent to activate a reporter gene in the presence of a competing DNA template lacking LexA binding sites. Furthermore, consistent with direct interaction with basal transcription factors, addition of recombinant TFIIF relieved squelching of basal transcription by AR142–485. Taken together these results suggest that one mechanism of transcriptional activation by the AR involves binding to TFIIF and recruitment of the transcriptional machinery.

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Strand-specific transcripts of a satellite DNA of the newts, Notophthalmus and Triturus, are present in cells in monomeric and multimeric sizes. These transcripts undergo self-catalyzed, site-specific cleavage in vitro: the reaction requires Mg2+ and is mediated by a “hammerhead” domain. Transcription of the newt ribozyme appears to be performed by RNA polymerase II under the control of a proximal sequence element and a distal sequence element. In vitro, the newt ribozyme can cleave in trans an RNA substrate, suggesting that in vivo it might be involved in RNA processing events, perhaps as a riboprotein complex. Here we show that the newt ribozyme is in fact present as a riboprotein particle of about 12 S in the oocytes of Triturus. In addition, reconstitution experiments and gel-shift analyses show that a complex is assembled in vitro on the monomeric ribozyme molecules. UV cross-linking studies identify a few polypeptide species, ranging from 31 to 65 kDa, associated to the newt ribozyme with different affinities. Finally, we find that an appropriate oligoribonucleotide substrate is specifically cleaved by the riboproteic activity in S-100 ovary extracts.

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Glutamic acid 286 (E286; Escherichia coli cytochrome bo3 numbering) in subunit I of the respiratory heme-copper oxidases is highly conserved and has been suggested to be involved in proton translocation. We report a technique of enzyme reconstitution that yields essentially unidirectionally oriented cytochrome bo3 vesicles in which proton translocation can be measured. Such experiments are not feasible in the E286Q mutant due to strong inhibition of respiration, but this is not the case for the mutants E286D and E286C. The reconstituted E286D mutant enzyme readily translocates protons whereas E286C does not. Loss of proton translocation in the D135N mutant, but not in D135E or D407N, also is verified using proteoliposomes. Stopped-flow experiments show that the peroxy intermediate accumulates in the reaction of the E286Q and E286C mutant enzymes with O2. We conclude that an acidic function of the 286 locus is essential for the mechanism of proton translocation.

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Human deoxyribonucleoside kinases are required for the pharmacological activity of several clinically important anticancer and antiviral nucleoside analogs. Human deoxycytidine kinase and thymidine kinase 1 are described as cytosolic enzymes in the literature, whereas human deoxyguanosine kinase and thymidine kinase 2 are believed to be located in the mitochondria. We expressed the four human deoxyribonucleoside kinases as fusion proteins with the green fluorescent protein to study their intracellular locations in vivo. Our data showed that the human deoxycytidine kinase is located in the cell nucleus and the human deoxyguanosine kinase is located in the mitochondria. The fusion proteins between green fluorescent protein and thymidine kinases 1 and 2 were both predominantly located in the cytosol. Site-directed mutagenesis of a putative nuclear targeting signal, identified in the primary structure of deoxycytidine kinase, completely abolished nuclear import of the protein. Reconstitution of a deoxycytidine kinase-deficient cell line with the wild-type nuclear or the mutant cytosolic enzymes both restored sensitivity toward anticancer nucleoside analogs. This paper reports that a deoxyribonucleoside kinase is located in the cell nucleus and we discuss the implications for deoxyribonucleotide synthesis and phosphorylation of nucleoside analogs.

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Ro09-0198 is a tetracyclic polypeptide of 19 amino acids that recognizes strictly the structure of phosphatidylethanolamine (PE) and forms a tight equimolar complex with PE on biological membranes. Using the cyclic peptide coupled with fluorescence-labeled streptavidin, we have analyzed the cell surface localization of PE in dividing Chinese hamster ovary cells. We found that PE was exposed on the cell surface specifically at the cleavage furrow during the late telophase of cytokinesis. PE was exposed on the cell surface only during the late telophase and no alteration in the distribution of the plasma membrane-bound cyclic peptide was observed during the cytokinesis, suggesting that the surface exposure of PE reflects the enhanced scrambling of PE at the cleavage furrow. Furthermore, cell surface immobilization of PE induced by adding the cyclic peptide coupled with streptavidin to prometaphase cells effectively blocked the cytokinesis at late telophase. The peptide-streptavidin complex treatment had no effect on furrowing, rearrangement of microtubules, and nuclear reconstitution, but specifically inhibited both actin filament disassembly at the cleavage furrow and subsequent membrane fusion. These results suggest that the redistribution of the plasma membrane phospholipids is a crucial step for cytokinesis and the cell surface PE may play a pivotal role in mediating a coordinate movement between the contractile ring and plasma membrane to achieve successful cell division.

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The objective of this study was to clarify the relative roles of medial versus luminal factors in the induction of thickening of the arterial intima after balloon angioplasty injury. Platelet-derived growth factor (PDGF) and thrombin, both associated with thrombosis, and basic fibroblast growth factor (bFGF), stored in the arterial wall, have been implicated in this process. To unequivocally isolate the media from luminally derived factors, we used a 20-μm thick hydrogel barrier that adhered firmly to the arterial wall to block thrombus deposition after balloon-induced injury of the carotid artery of the rat. Thrombosis, bFGF mobilization, medial repopulation, and intimal thickening were measured. Blockade of postinjury arterial contact with blood prevented thrombosis and dramatically inhibited both intimal thickening and endogenous bFGF mobilization. By blocking blood contact on the two time scales of thrombosis and of intimal thickening, and by using local protein release to probe, by reconstitution, the individual roles of PDGF-BB and thrombin, we were able to conclude that a luminally derived factor other than PDGF or thrombin is required for the initiation of cellular events leading to intimal thickening after balloon injury in the rat. We further conclude that a luminally derived factor is required for mobilization of medial bFGF.

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Cellular proliferation and tissue remodeling are central to the regenerative response after a toxic injury to the liver. To explore the role of plasminogen in hepatic tissue remodeling and regeneration, we used carbon tetrachloride to induce an acute liver injury in plasminogen-deficient (Plgo) mice and nontransgenic littermates (Plg+). On day 2 after CCl4, livers of Plg+ and Plgo mice had a similar diseased pale/lacy appearance, followed by restoration of normal appearance in Plg+ livers by day 7. In contrast, Plgo livers remained diseased for as long as 2.5 months, with a diffuse pale/lacy appearance and persistent damage to centrilobular hepatocytes. The persistent centrilobular lesions were not a consequence of impaired proliferative response in Plgo mice. Notably, fibrin deposition was a prominent feature in diseased centrilobular areas in Plgo livers for at least 30 days after injury. Nonetheless, the genetically superimposed loss of the Aα fibrinogen chain (Plgo/Fibo mice) did not correct the abnormal phenotype. These data show that plasminogen deficiency impedes the clearance of necrotic tissue from a diseased hepatic microenvironment and the subsequent reconstitution of normal liver architecture in a fashion that is unrelated to circulating fibrinogen.

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Our model of the native fatty acid synthase (FAS) depicts it as a dimer of two identical multifunctional proteins (Mr ≈ 272,000) arranged in an antiparallel configuration so that the active Cys-SH of the β-ketoacyl synthase of one subunit (where the acyl group is attached) is juxtaposed within 2 Å of the pantetheinyl-SH of the second subunit (where the malonyl group is bound). This arrangement generates two active centers for fatty acid synthesis and predicts that if we have two appropriate halves of the monomer, we should be able to reconstitute an active fatty acid-synthesizing site. We cloned, expressed, and purified catalytically active thioredoxin (TRX) fusion proteins of the NH2-terminal half of the human FAS subunit protein (TRX-hFAS-dI; residues 1–1,297; Mr ≈ 166) and of the C-terminal half (TRX-hFAS-dII-III; residues 1,296–2,504; Mr ≈ 155). Adding equivalent amounts of TRX-hFAS-dI and TRX-hFAS-dII-III to a reaction mixture containing acetyl-CoA, malonyl-CoA, and NADPH resulted in the synthesis of long-chain fatty acids. The rate of synthesis was dependent upon the presence of both recombinant proteins and reached a constant level when they were present in equivalent amounts, indicating that the reconstitution of an active fatty acid-synthesizing site required the presence of every partial activity associated with the subunit protein. Analyses of the product acids revealed myristate to be the most abundant with small amounts of palmitate and stearate, possibly because of the way the fused recombinant proteins interacted with each other so that the thioesterase hydrolyzed the acyl group in its myristoyl state. The successful reconstitution of the human FAS activity from its domain I and domains II and III fully supports our model for the structure–function relationship of FAS in animal tissues.

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The final step in glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins consists of a transamidation reaction in which preassembled GPI donors are substituted for C-terminal signal sequences in nascent polypeptides. In previous studies we described a human K562 cell mutant, termed class K, that accumulates fully assembled GPI units but is unable to transfer them to N-terminally processed proproteins. In further work we showed that, unlike wild-type microsomes, microsomes from these cells are unable to support C-terminal interaction of proproteins with the small nucleophiles hydrazine or hydroxylamine, and that the cells thus are defective in transamidation. In this study, using a modified recombinant vaccinia transient transfection system in conjunction with a composite cDNA prepared by 5′ extension of an existing GenBank sequence, we found that the genetic element affected in these cells corresponds to the human homolog of yGPI8, a gene affected in a yeast mutant strain exhibiting similar accumulation of GPI donors without transfer. hGPI8 gives rise to mRNAs of 1.6 and 1.9 kb, both encoding a protein of 395 amino acids that varies in cells with their ability to couple GPIs to proteins. The gene spans ≈25 kb of DNA on chromosome 1. Reconstitution of class K cells with hGPI8 abolishes their accumulation of GPI precursors and restores C-terminal processing of GPI-anchored proteins. Also, hGPI8 restores the ability of microsomes from the mutant cells to yield an active carbonyl in the presence of a proprotein which is considered to be an intermediate in catalysis by a transamidase.

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We report herein the successful long term engraftment of highly purified hematopoietic stem cells (HSCs) without any facilitating cells in fully allogeneic recipient mice across the entire major histocompatibility complex (MHC) transplantation barrier. This finding challenges the assumption that highly purified marrow HSCs alone cannot produce long-lived allogeneic bone marrow chimeras across the MHC barrier. In the present experiments, 1 × 105 HSCs from 5-fluorouracil (5-FU)-treated donors, without any facilitating cells, have been found to repopulate lethally irradiated fully allogeneic recipients. Low density, lineage-negative (CD4−, CD8−, B220−, Mac-1−, Gr-1−), CD71-negative, class I highly positive, FACS-sorted cells from 5-FU-treated C57BL/6 (B6) donor mice were transplanted into lethally irradiated BALB/c recipients. (BALB/c → BALB/c) → BALB/c T cell-depleted marrow cells used as compromised cells were also transplanted into the recipients to permit experiments to be pursued over a long period of time. Cells of donor origin in all recognized lineages of hematopoietic cells developed in these allogeneic chimeras. One thousand HSCs were sufficient to repopulate hemiallogeneic recipients, but 1 × 104 HSCs alone from 5-FU-treated donors failed to repopulate the fully allogeneic recipients. Transplantation of primary marrow stromal cells or bones of the donor strain into recipient, together with 1 × 104 HSCs, also failed to reconstitute fully allogeneic recipients. Suppression of resistance of recipients by thymectomy or injections of granulocyte colony-stimulating factor before stem cell transplantation enhanced the engraftment of allogeneic HSCs. Our experiments show that reconstitution of all lymphohematopoietic lineages across the entire MHC transplantation barriers may be achieved by transplanting allogeneic HSCs alone, without any facilitating cells, as long as a sufficient number of HSCs is transplanted.

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A strategy for cloning and mutagenesis of an infectious herpesvirus genome is described. The mouse cytomegalovirus genome was cloned and maintained as a 230 kb bacterial artificial chromosome (BAC) in E. coli. Transfection of the BAC plasmid into eukaryotic cells led to a productive virus infection. The feasibility to introduce targeted mutations into the BAC cloned virus genome was shown by mutation of the immediate-early 1 gene and generation of a mutant virus. Thus, the complete construction of a mutant herpesvirus genome can now be carried out in a controlled manner prior to the reconstitution of infectious progeny. The described approach should be generally applicable to the mutagenesis of genomes of other large DNA viruses.

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The split-ubiquitin technique was used to detect transient protein interactions in living cells. Nub, the N-terminal half of ubiquitin (Ub), was fused to Sec62p, a component of the protein translocation machinery in the endoplasmic reticulum of Saccharomyces cerevisiae. Cub, the C-terminal half of Ub, was fused to the C terminus of a signal sequence. The reconstitution of a quasi-native Ub structure from the two halves of Ub, and the resulting cleavage by Ub-specific proteases at the C terminus of Cub, serve as a gauge of proximity between the two test proteins linked to Nub and Cub. Using this assay, we show that Sec62p is spatially close to the signal sequence of the prepro-α-factor in vivo. This proximity is confined to the nascent polypeptide chain immediately following the signal sequence. In addition, the extent of proximity depends on the nature of the signal sequence. Cub fusions that bore the signal sequence of invertase resulted in a much lower Ub reconstitution with Nub-Sec62p than otherwise identical test proteins bearing the signal sequence of prepro-α-factor. An inactive derivative of Sec62p failed to interact with signal sequences in this assay. These in vivo findings are consistent with Sec62p being part of a signal sequence-binding complex.

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Assembly and modulation of focal adhesions during dynamic adhesive processes are poorly understood. We describe here the use of ventral plasma membranes from adherent fibroblasts to explore mechanisms regulating integrin distribution and function in a system that preserves the integration of these receptors into the plasma membrane. We find that partial disruption of the cellular organization responsible for the maintenance of organized adhesive sites allows modulation of integrin distribution by divalent cations. High Ca2+ concentrations induce quasi-reversible diffusion of β1 integrins out of focal adhesions, whereas low Ca2+ concentrations induce irreversible recruitment of β1 receptors along extracellular matrix fibrils, as shown by immunofluorescence and electron microscopy. Both effects are independent from the presence of actin stress fibers in this system. Experiments with cells expressing truncated β1 receptors show that the cytoplasmic portion of β1 is required for low Ca2+-induced recruitment of the receptors to matrix fibrils. Analysis with function-modulating antibodies indicates that divalent cation-mediated receptor distribution within the membrane correlates with changes in the functional state of the receptors. Moreover, reconstitution experiments show that purified α-actinin colocalizes and redistributes with β1 receptors on ventral plasma membranes depleted of actin, implicating binding of α-actinin to the receptors. Finally, we found that recruitment of exogenous actin is specifically restricted to focal adhesions under conditions in which new actin polymerization is inhibited. Our data show that the described system can be exploited to investigate the mechanisms of integrin function in an experimental setup that permits receptor redistribution. The possibility to uncouple, under cell-free conditions, events involved in focal adhesion and actin cytoskeleton assembly should facilitate the comprehension of the underlying molecular mechanisms.