The CREA repressor responsible for carbon catabolite repression in Aspergillus nidulans represses the transcription of the ethanol regulon. The N-terminal part of the CREA protein encompassing the two zinc fingers (C2H2 class family) and an alanine-rich region was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase. Our results show that CREA is a DNA-binding protein able to bind to the promoters of both the specific trans-acting gene, alcR, and of the structural gene, alcA, encoding the alcohol dehydrogenase I. DNase I protection footprinting experiments revealed several specific binding sites in the alcR and in the alcA promoters having the consensus sequence 5'-G/CPyGGGG-3'. The disruption of one of these CREA-binding sites in the alcR promoter overlapping the induction target for the trans-activator ALCR results in a partially derepressed alc phenotype and derepressed alcR transcription, showing that this binding site is functional in vivo. Our data suggest that CREA represses the ethanol regulon by a double lock mechanism repressing both the trans-acting gene, alcR, and the structural gene, alcA.
The ALCR protein is the transcriptional activator of the ethanol utilization pathway in the filamentous fungus AspergiUlus nidulans. This activator belongs to a family of fungal proteins having a conserved DNA-binding domain containing six cysteines (C6 class) with some striking features. At variance with other motifs of this class, the binding domain of ALCR is strongly asymmetrical in relation to the central cysteines and moreover was predicted to adopt a helix-turn-helix structure. This domain of ALCR was synthesized in Escherichia coli and purified as a glutathione-S-transferase fusion protein. Our results show that the transcriptional activator ALCR is a DNA-binding protein. The DNA-binding motif contains zinc that is necessary for the specific DNA binding. The ALCR peptide binds upstream of the coding region of alcR to two specific targets with different affinities that are characterized by a conserved 5-nucleotide core, 5'-CCGCA-3' (or its reverse). One site, the lower-affinity binding site, is a direct repeat, and the other, the higher-affinity binding site, is a palindromic sequence with dyad symmetry. Therefore, the ALCR binding protein is able to recognize one DNA sequence in two different configurations. An alcR mutant obtained by deletion of the two specific targets in the cis-acting region of the alcR gene is unable to grow on ethanol and does not express any alcohol dehydrogenase activity. These results demonstrate that the binding sites are in vivo functional targets (UASaic) for the ALCR protein in A. nidulans. They corroborate prior evidence that alcR is autoregulated.Positive control mechanisms in eukaryotes have been extensively characterized. They are mediated by transcription factors that bind to specific DNA targets. The expression of genes encoding the ethanol utilization enzymes in the ascomycete Aspergillus nidulans is regulated by the pathway-specific transactivator ALCR. In conditions of induction (by ethanol or gratuitous inducers like ethylmethylketone), the ALCR protein is necessary for the expression of the two structural genes alcA, encoding alcohol dehydrogenase I, and aldA, encoding aldehyde dehydrogenase (30, 36). Transcription of these two genes can be strongly induced, and this property was widely used for the expression of heterologous proteins (for a review, see reference 11). The expression of the alcR gene is inducible, positively autoregulated, and subjected to carbon catabolite repression under the control of the negatively acting gene creA (11,25,30). The three genes of the ethanol regulon were cloned and sequenced (12,16,26,32), and the creA gene identified by Bailey and Arst (2) was also cloned (8) and sequenced (9). The transcription factor ALCR is 821 amino acids long (12) and contains a sequence of six Cys residues, Cys-X2-Cys-X6-Cys-X16-Cys-X2-Cys-X6-Cys within its N-terminal part. It is related to the highly conserved DNA-binding domain of the transcription factors of the C6 class of the ascomycetes (23). At variance with other motifs of this class, the putativ...
We have demonstrated recently that Birbeck granule-positive Langerhans cells (LC) can be derived from CD34+ peripheral blood progenitor cells in the presence of a seven-cytokine cocktail (CC7–7). Here, we show that the sequential use of early-acting hematopoietic growth factors, stem cell factor, interleukin (IL)-3, and IL-6, followed on day 8 by differentiation in the two-factor combination IL-4 plus granulocytemacrophage colony-stimulating factor (GM-CSF) (CC4GM) is more efficient and allows the cells to be arrested in the LC stage for more than 1 week while continuous maturation occurs in CC7–7. Maturation of LC to interdigitating dendritic cells (DC) could specifically be induced within 60 hours by addition of tumor necrosis factor-alpha (20 ng/mL) or lipopolysaccharide (100 ng/mL). Using LC that had been enriched to greater than 90% CD1a+ cells by an immunoaffinity column, we were able to define clear-cut differences between LC and DC that corroborate data of the respective cells derived from epithelial borders (LC) or from lymph nodes (LN) and spleen (DC). Thus, molecules and functions involved in antigen (AG) uptake and processing were highly expressed in LC, while those involved in AG presentation were at maximum in DC. LC were CD1a+2 DR+2, CD23+, CD36+, CD80-, CD86-, and CD25-, while DC were CD1a+/- DR+3, CD23-, CD36-, CD80+, CD86+2, and CD25+, CD40 and CD32 were moderately expressed and nearly unchanged on maturation, in contrast to monocyte-derived DC. Macropinocytosis of fluorescein isothiocyanate-dextran was dominant in LC, as were multilamellar major histocompatibility complex (MHC) class II compartments (MIICs), which were detected by electron microscopy. The functional dichotomy of these cell types was finally supported by testing the AG-presenting cell function for tetanus toxoid to primed autologous T-cell lines, which was optimal when cells were loaded with AG as LC and subsequently induced to become DC.
Diploid fibroblast (dFb) cultures were established from a total of 106 skin and serosa biopsies of human adults. Using an optimized enzymatic dissociation procedure, 10(11) dFb/cm2 skin were obtained from patients younger than 60 years after an average time of 89 +/- 8 days, with a mean population doubling time of 3.87 +/- 1.4 days. Enzymatic dissociation of skin biopsies yielded cultures of significantly higher growth capacity of dFb than those prepared by mechanical dissociation followed by spontaneous outgrowth of cells. The plating efficiency that may be crucial for clonal selection of transfected cells was negligible when dFb were plated without feeder cells at low density, while it was enhanced to 9-24% by the addition of a feeder layer of irradiated human embryonal fibroblasts. DFb secreted various cytokines with spontaneous release of interleukin-6 (IL-6) in high quantities of up to 20 ng/10(6) cells/24 hr. In addition, one-third of the culture secreted substantial amounts of granulocyte-macrophage colony-stimulating factor (GM-CSF), while low amounts of tumor necrosis factor-alpha (TNF-alpha) were detectable in some cases after irradiation of the cells. Comparison of various transfection methods by a transient luciferase expression assay demonstrated that receptor-mediated gene transfer was approximately 10-fold more efficient than cationic lipofection of dFb, while electroporation resulted in substantially less expression of the reporter gene. We conclude that primary dFb can be obtained reproducibly from human adults and represent a suitable target cell population for receptor-mediated gene transfer and cationic lipofection.(ABSTRACT TRUNCATED AT 250 WORDS)
Migration of leukocytes to injured tissues is a hallmark of inflammation. The recruitment phase of cells can be subdivided into three steps: the rolling phase, the firm adhesion phase, and the transendothelial migration phase. Each step is mediated by a complex interplay of endothelial/leukocyte surface molecule interactions (mostly of selectin and integrin families) as well as a group of small, secreted peptides, called chemokines. Chemokines activate on the one hand, the leukocytes to express the appropriate adhesion molecules and on the other hand, they lead to transendothelial migration via chemotaxis (migration along a gradient in solution) and haptotaxis (migration along a gradient bound to extracellular martrices or cell membranes). The structure, biology and pathobiology of the more than 22 known members of this group of soluble mediators, with a particular emphasis on their past and present nomenclature, is the topic of this minireview.
SummaryIn an attempt to characterize genes participating in the allergic late phase reaction, we have isolated a novel intercrine/chemokine (called MARC) from a cDNA library of the stimulated mouse mast cell line, CPlI. As measured by Northern blotting, it is strongly upregulated at the mRNA level after the physiological challenge of the cells with immunoglobulin (Ig)E plus antigen. Unstimulated cells completely lack significant, stable expression, as do a number of other, different cell lines (uninduced and induced) and mouse tissues. In contrast to the Northern blot analysis, a polymerase chain reaction (PCR) analysis, performed on CPII ceUs and on Perco11 gradient purified mouse peritoneal mast cells, revealed a basal level of transcription in the uninduced stage. After 2 h of IgE plus antigen challenge, a quantitative reverse transcriptase-PCR, using a spiked in MIMIC, showed a level of transcripts more than 100-fold higher in the CPII cells and 5-20-fold higher in purified mouse peritoneal cavity mast cells. This rapid induction after the Fc+RI challenge, the identification of the gene as a member of the chemokine family, and its upregulated expression in peritoneal mast cells, all suggest an involvement in certain acute and chronic pathological mast cell-driven diseases.I ntercrines/r are small soluble proteins that regulate the physiological tra~cking and the partial activation ofleukocytes (1). In contrast to most other known cytokines and lymphokines, chemokines show a considerable homology (identity and similarity) at the amino acid level Additionally, they are characterized by a common protein structure of two loops formed via disulfide bridges of four highly conserved cysteines. Based on the location of the two NH2-terminal cysteines, this superfamily is subdivided into a CXC (IL-8 or PF4 family) and CC (RANTES/sis family) branch (2, 3). In the last, six members in the human system and five corresponding mouse genes are currently isolated (4, 5).Spedfic sites of production in the body and the low amount produced in vivo make the direct isolation and characterization of these proteins from healthy individuals nearly impossible. A few members, like macrophage inflammatory protein (MIP)-la, MIP-IB and monocyte chemotactic protein (MCP)I, have been purified at protein level from overexpressing tumor cell lines by functional monitoring, and the corresponding genes were subsequently identified (6, 7). However, the majority in the CC branch were isolated by inductionspecific differential hybridization of cDNA libraries (see reference 4). This reflects the fact that most members are strongly upregulated at the transcriptional level after cell activation (4). Supernatants from transiently transfected cell lines were used for a detailed functional analysis afterwards (8).Type I allergic reactions are characterized at the level of mast cells by a biphasic response. In an immediate reaction (up to several minutes) preformed low molecular weight substances, like histamine and serotonin, are released after IgE plu...
It is well established by in vivo and in vitro studies that dendritic cells (DCs) originate from hematopoietic progenitor cells. However, the presumed intermediate of Birbeck granule (BG)+ Langerhans cells (LCs) has not been detected in cultures derived from bone marrow or peripheral blood progenitor cells (PBPCs), thus contrasting with the data obtained with cord blood. We show here that large numbers of BG+ LCs can be generated from human CD34+ PBPCs in vitro, when granulocyte-macrophage colony-stimulating factor and interleukin-4, potent promotors of LC/DC differentiation, are combined with a cocktail of early acting hematopoietic growth factors. LCs were found to emerge from CD33+CD11b+CD14-progenitor cells that they share with the monocytic lineage. During culture, these cells exhibited a sequence of dramatic morphologic changes, starting with a major increase in granularity followed by an increase in size herein exceeding that of all peripheral blood cells. At the same time, CD1a and major histocompatibility complex class II expression were upregulated and virtually all CD1a++ cells were BG+ by electron microscopy. With prolonged culture, CD1a was downregulated on a major population of cells, paralleled by a loss of BG and an increase of CD4, CD25, and CD80 expression that may correspond to the maturation of epidermal LC in vitro. However, these cells were consistently CD5- and did not exhibit changes in the CD45-isoform expression during culture. The availability of large numbers of these highly purified BG+ LCs and mature DCs allows for specific analysis of these subpopulations and provides a source of potent antigen-presenting cells from individual patients for vaccination protocols against infectious or tumor-associated antigens.
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