Regulation of intercellular adhesion molecule-1 gene by tumor necrosis factor-alpha is mediated by the nuclear factor-kappaB heterodimers p65/p65 and p65/c-Rel in the absence of p50.

Human intercellular adhesion molecule-1 (ICAM-1) plays an important role in immune responses as the major specific ligand for the beta2-integrins LFA-1 and Mac-1. During the inflammatory process, ICAM-1 expression is stimulated by various proinflammatory cytokines. We have examined the mechanisms of transcriptional control involved in the stimulation of ICAM-1 gene expression by tumor necrosis factor-alpha (TNF-alpha) and by the nuclear factor-kappaB (NF-kappaB) family of transcription factors in the Ad5-transformed human embryonal kidney cell line 293. A proximal site (5'-TTGGAAATTCC-3') mapping at position -228 from the ATG and known to mediate TNF-alpha responsiveness in endothelial cells is also critical for TNF-alpha responsiveness in 293 cells. However, unlike endothelial cells, electrophoretic mobility shift assays, using whole-cell extracts prepared from TNF-alpha-treated cells, showed that TNF-alpha induces the formation of a specific kappaB binding complex, mainly composed of NF-kappaB subunits RelA and c-Rel. Electrophoretic mobility shift assays done with 293 cells transfected with p50, p65, or both subunits showed that p50 only has a weak ability to bind the proximal ICAM-1 NF-kappaB site. Another element exhibiting sequence homology with NF-kappaB binding sites and located at position -540 relative to the mRNA cap site was found to be involved in the basal activity of the ICAM-1 promoter, is not required for TNF-alpha responsiveness, and does not bind NF-kappaB subunits. Whereas transactivation of the ICAM-1 promoter by p65 requires the proximal NF-kappaB site, deletion mutant analysis showed that p50 and, to a greater extent, p52 transactivate reporter plasmids lacking NF-kappaB sites, suggesting the presence of other p50/p52 responsive element(s).

Heterodimeric retinoic acid receptor-beta and retinoid X receptor-alpha complexes stimulate expression of the intercellular adhesion molecule-1 gene.

Human intercellular adhesion molecule-1 (ICAM-1), a specific ligand for the leukocyte-function associated antigen-1 and for Mac-1, plays an important role in immune responses. ICAM-1 expression is regulated by various proinflammatory cytokines, phorbol myristate acetate, and retinoic acid. In this study, we investigated the mechanisms of transcriptional control involved in the stimulation of ICAM-1 gene expression by retinoic acid in Cos-1 cells. Deletion mutant analysis provided evidence that a region located between -393 and -176 from the translational start site is critical to retinoic acid stimulation of luciferase activity. This region harbors the consensus sequence for a retinoic acid-responsive element (RARE) 5'-GGGTCATCGCCCTGCCA-3'. The Smal(-270)/Smal (-178) fragment containing this element conferred appropriate retinoic acid responsiveness to an enhancerless SV40 promoter. Cotransfection of expression vectors encoding the retinoic acid receptor alpha, beta, or gamma and retinoid X receptor alpha with reporter plasmids harboring the putative RARE demonstrated that the ICAM-1 gene is regulated by retinoic acid in a retinoic acid receptor beta/retinoid X receptor alpha-dependent fashion. Electrophoretic mobility shift assays showed that ICAM-1 and ADH3 RARE, a well-characterized RARE, display the same band shift pattern, bind retinoic acid receptor beta and retinoid X receptor alpha, and are mutually competitive.

Modes of action and inhibitory activities of new siderophore-beta-lactam conjugates that use specific iron uptake pathways for entry into bacteria.

We describe here the mechanism of inhibition of two new siderophore-beta-lactam conjugates against Escherichia coli X580. One conjugate is a spermidine-based catechol siderophore-carbacephalosporin (JAM-2-263), and the other is an N5-acetyl-N5-hydroxy-L-ornithine tripeptide hydroxamate siderophore-carbacephalosporin (EKD-3-88). In an agar diffusion test, both conjugates produced large inhibitory zones against strain X580. Resistant strains (i.e., JAMR and EKDR) could be isolated after exposure of X580 to the conjugates JAM-2-263 and EKD-3-88, respectively. No cross-resistance was observed in these individual isolates. JAMR and EKDR were studied further to elucidate the mechanism of inhibition of each conjugated drug. The affinities of JAM-2-263 and EKD-3-88 for penicillin-binding proteins (PBPs) of isolated inner membranes were determined by a competition assay with 125I-penicillin V. JAM-2-263 targeted primarily PBPs 1A/B and 5/6, while EKD-3-88 targeted PBPs 1A/B and 3. Strains X580, JAMR, and EKDR showed similar PBP affinities for the conjugates. However, marked changes were observed in the iron-regulated outer membrane proteins of resistant isolates grown on agar plates depleted of iron. EKDR lost the expression of FhuA (78 kDa) and its sensitivity to phages T1 and T5, whereas JAMR lost the expression of Cir (74 kDa) and its sensitivity to colicin Ia. These results revealed the requirement of FhuA and Cir for the inhibitory activities of EKD-3-88 and JAM-2-263, respectively. In an antibiotic diffusion assay, ferrichrome (1 microM) strongly antagonized the activities of both conjugates against X580 and JAMR, including the residual activity of JAM-2-263 against JAMR. However, the susceptibility of strain EKDR lacking the ferrichrome receptor (FhuA-) to the two conjugates remained the same in the presence of ferrichrome. The antagonistic effect of ferrichrome on the activity of JAM-2-263 may also indicate a role for FhuA in the activity of this beta-lactam conjugate. A FhuA- Cir- double mutant confirmed this hypothesis, since it showed a higher level of resistance to JAM-2-263. To reproduce iron-restricted in vivo growth conditions, we grew X580 and EKDR cells in diffusion chambers implanted in the peritoneal cavities of rats. Strain EKDR showed impaired growth in such a cultivation system. This is the first report of beta-lactam drug transport into E. coli cells that involves the FhuA outer membrane protein.

Influence of growth media on Escherichia coli cell composition and ceftazidime susceptibility.

Cell composition and surface properties of Escherichia coli were modified by using various growth media to investigate the role of yet uncharacterized components in ceftazidime susceptibility. An eightfold dilution of Luria broth was used as the basic growth medium and was supplemented with up to 4% phosphate, 5% glucose, or 12% L-glutamate. Decreases in cephaloridine and ceftazidime susceptibility, of two- and eightfold, respectively, were observed only in the glucose-enriched medium. The outer membrane permeability to ceftazidime and cephaloridine was evaluated by crypticity indices. Indices were unchanged under all growth conditions. Fluorometry of whole cells with 1-N-phenylnaphthylamine showed that glucose does not affect the interaction of this hydrophobic probe with the membranes but showed that elevated concentrations of phosphate or glutamate cause a marked increase in cell hydrophobicity, which, in turn, correlates with an increase in the susceptibility of E. coli to nalidixic acid. Growth in phosphate- or glutamate-enriched media caused an augmentation in major phospholipid species and may explain the increased hydrophobicity and susceptibility of E. coli to nalidixic acid. These data showed that E. coli susceptibility to ceftazidime is not influenced by cell surface hydrophobicity and suggested that the contribution of a nonspecific lipophilic diffusion route for entry of ceftazidime into cells is not likely to occur or is distinct from that of more hydrophobic molecules such as nalidixic acid. Finally, the penicillin-binding proteins of the E. coli cells were also investigated. Penicillin-binding protein 8 was only markedly labeled with 125I-penicillin V in inner membranes extracted from cells grown with glucose. Results of this study suggest that the unexpected change in penicillin-binding protein 8 observed in the presence of glucose may be responsible for the increase in MICs of cephaloridine and ceftazidime.