A four-dimensional study of the aortic root dynamics.

OBJECTIVE: Although aortic root expansion has been well studied, its deformation and physiologic relevance remain controversial. Three-dimensional (3-D) sonomicrometry (200Hz) has made time-related 4-D study possible. METHODS: Fifteen sonomicrometric crystals were implanted into the aortic root of eight sheep at each base (three), commissures (three), sinuses of Valsalva (three), sinotubular junction (three), and ascending aorta (three). In this acute, open-chest model, the aortic root geometric deformations were time related to left ventricular and aortic pressures. RESULTS: During the cardiac cycle, aortic root volume increased by mean+/-1 standard error of the mean (SEM) 33.7+/-2.7%, with 36.7+/-3.3% occurring prior to ejection. Expansion started during isovolumic contraction at the base and commissures followed (after a delay) by the sinotubular junction. At the same time, ascending aorta area decreased (-2.6+/-0.4%). During the first third of ejection, the aortic root reached maximal expansion followed by a slow, then late rapid decrease in volume until mid-diastole. During end-diastole, the aortic root volume re-expanded by 11.3+/-2.4%, but with different dynamics at each area level. Although the base and commissural areas re-expanded, the sinotubular junction and ascending aorta areas kept decreasing. At end-diastole, the aortic root had a truncated cone shape (base area>commissures area by 51.6+/-2.0%). During systole, the root became more cylindrical (base area>commissures area by 39.2+/-2.5%) because most of the significant changes occurred at commissural level (63.7+/-3.6%). CONCLUSION: Aortic root expansion follows a precise chronology during systole and becomes more cylindrical - probably to maximize ejection. These findings might stimulate a more physiologic approach to aortic valve and aortic root surgical procedures.

Aortic and pulmonary root: are their dynamics similar?

OBJECTIVES: The long-term behavior of the pulmonary autograft in the aortic position (Ross procedure) remains uncertain. Using three-dimensional (3D) sonomicrometry (200 Hz) we compared the dynamics of the aortic and pulmonary roots. METHODS: Twenty-four crystals were implanted in each aortic (eight sheep) and pulmonary roots (six sheep) at: base (3 x 2), commissures (3 x 2), sinotubular junction (3 x 2), ascending aorta (3) and pulmonary trunk (3). Under stable hemodynamic conditions, geometric changes were time-related to left ventricular pressure (LV) and aortic pressure. RESULTS: The expansion of the aortic root is twice that of the pulmonary root. During the cardiac cycle, the aortic root volume increased by 37.7 +/- 2.7% (mean +/- SEM) versus 20.9 +/- 1.0% for the pulmonary root. Both were cone-shaped at end diastole. Because expansion at commissures was twice that of the base, both roots became more cylindrical during ejection. Although both roots started to expand prior to ejection and reached maximal expansion during the first third of ejection, the commissural and sinotubular junction dynamics were different in each root. While in the aortic root, expansion at commissural and sinotubular junction levels was significantly different (63.7 +/- 3.6% versus 37.0 +/- 2.1%), in the pulmonary root, they were similar (29.0 +/- 1.3% versus 27.7 +/- 1.4%). Expansion of the three sinuses was also different (P<0.001). In the aortic root: the right expanded more than the left and more than the non-coronary sinus. In the pulmonary root: the right sinus expanded more than the anterior more than the left. CONCLUSIONS: Dynamic differences might explain the global pulmonary root dilatation when subjected to systemic pressure, particularly at the level of the sinotubular junction which might result in the autograft failure. Differences in the asymmetrical expansion of the aortic and pulmonary roots should be considered for the implantation of the pulmonary autograft in the most physiological position.

A dynamic anatomical study of ulnar nerve motion after anterior transposition for cubital tunnel syndrome.

Cubital tunnel syndrome is the second most commonly encountered compression neuropathy of the upper limb. Multiple techniques for surgical management have been proposed but no universally accepted algorithm for management exists. Six cadaveric upper limbs underwent ulnar nerve decompression and anterior transposition into subcutaneous and then submuscular positions. After marking nerves with tungsten, radiological examination of nerve motion was performed and nerve angulations were measured in the region of the flexor carpi ulnaris (FCU) origin. Comparison of ulnar nerves in each position revealed statistically significant greater angulation after subcutaneous transposition than after submuscular transposition with the elbow held in full flexion. This point of angulation may act as a secondary point of compression or as a focus for neuritis and scar formation. This finding can contribute to the understanding of why differing outcomes may be observed after different forms of anterior transposition.

Rapid lipopolysaccharide-induced differentiation of CD14(+) monocytes into CD83(+) dendritic cells is modulated under serum-free conditions by exogenously added IFN-gamma and endogenously produced IL-10.

We showed previously that about half of purified CD14(+) peripheral blood monocytes cultured under serum-free conditions and treated with GM-CSF and bacterial LPS rapidly (2 - 4 day) differentiate into CD83(+) dendritic cells (DC). The remaining cells retain the CD14(+)/CD83(-) monocyte/macrophage phenotype. In order to identify factors that influence whether monocytes differentiate into DC or remain on the monocyte/macrophage developmental pathway, we evaluated the effects of exogenously added IFN-gamma and endogenously produced IL-10 on the proportion and function of CD14(+) monocytes that adopt DC characteristics in response to LPS. IFN-gamma priming dramatically increased the proportion of monocytes that adopted stable DC characteristics in response to LPS, improved their T cell allosensitizing capacity, and enhanced levels of secreted IL-12 heterodimer. IFN-gamma priming also suppressed the production of IL-10, a cytokine known to have inhibitory effects on DC differentiation. When monocytes were treated with LPS plus IL-10-neutralizing antibodies, dramatically enhanced DC differentiation, IL-12 secretion, and T cell allosensitizing capacity were observed, mimicking in many respects the effects of IFN-gamma priming. IFN-gamma primed cells still displayed appreciable sensitivity to exogenously added IL-10, suggesting that attenuated IL-10 secretion is partially responsible for the enhancing effects of IFN-gamma. These studies therefore identify IFN-gamma as a DC differentiation co-factor for CD14(+) monocytes, and IL-10 as an autocrine/paracrine inhibitor of DC differentiation, linking these agents for the first time as mutually opposed regulators that govern whether CD14(+) cells differentiate into DC upon contact with LPS or remain on the monocyte/macrophage developmental pathway.

CD14+ monocytes as dendritic cell precursors: diverse maturation-inducing pathways lead to common activation of NF-kappab/RelB.

Dendritic cells are extremely potent antigen-presenting cells that are primarily responsible for the sensitization of naïve T cells to protein antigen in vivo. For this reason, dendritic cells are the focus of intense study. Despite this interest, relatively little information is available on the signal transduction pathways that regulate the development and activity of these cells. The last several years, however, have seen a steady accumulation of data regarding methods to cultivate large numbers of DC, the characterization of attendant signals that drive DC development from various precursor cells, and the induction of nuclear transcription factors that presumably direct alterations in gene expression that regulate aspects of DC development. In this review, we briefly summarize some of these findings, with emphasis on monocyte-derived dendritic cells and a discussion of two distinct types of signaling pathways that appear to regulate the final maturation of DC: one pathway calcium-dependent and cyclosporine A-sensitive, the other pathway CsA-insensitive. Although evidence suggests these signaling pathways are quite divergent in their upstream components, they both appear to activate NF-kappaB nuclear factors, particularly RelB.

Capitation and risk adjustment in health care financing: an international progress report.

In every system of health care, capitation payments have become the accepted tool used by health care purchasers in much of the developed world to determine prospective budgets. The policy prescription of capitation is perceived to address both equity objectives (of great importance in publicly funded systems of health care) and efficiency objectives (the dominant concern in competitive insurance markets). An examination of the current state of the art in 20 countries outside the United States in which health care capitation has been implemented confirms that capitation has assumed central importance within diverse systems of health care. In practice, however, the setting of capitation payments has been heavily constrained to date by poor data availability and unsatisfactory analytic methodology.

Bacterial lipopolysaccharide, TNF-alpha, and calcium ionophore under serum-free conditions promote rapid dendritic cell-like differentiation in CD14+ monocytes through distinct pathways that activate NK-kappa B.

To facilitate the study of signaling pathways involved in myeloid dendritic cell (DC) differentiation, we have developed a serum-free culture system in which human CD14+ peripheral blood monocytes differentiate rapidly in response to bacterial LPS, TNF-alpha, or calcium ionophore (CI). Within 48-96 h, depending on the inducing agent, the cells acquire many immunophenotypical, morphological, functional, and molecular properties of DC. However, there are significant differences in the signaling pathways used by these agents, because 1) LPS-induced, but not CI-induced, DC differentiation required TNF-alpha production; and 2) cyclosporin A inhibited differentiation induced by CI, but not that induced by LPS. Nevertheless, all three inducing agents activated members of the NF-kappaB family of transcription factors, including RelB, suggesting that despite differences in upstream elements, the signaling pathways all involve NF-kappaB. In this report we also demonstrate and offer an explanation for two observed forms of the RelB protein and show that RelB can be induced in myeloid cells, either directly or indirectly, through a calcium-dependent and cyclosporin A-sensitive pathway.

Role of NF-kappaB in p53-mediated programmed cell death.

The tumour suppressor p53 inhibits cell growth through activation of cell-cycle arrest and apoptosis, and most cancers have either mutation within the p53 gene or defects in the ability to induce p53. Activation or re-introduction of p53 induces apoptosis in many tumour cells and may provide effective cancer therapy. One of the key proteins that modulates the apoptotic response is NF-kappaB, a transcription factor that can protect or contribute to apoptosis. Here we show that induction of p53 causes an activation of NF-kappaB that correlates with the ability of p53 to induce apoptosis. Inhibition or loss of NF-kappaB activity abrogated p53-induced apoptosis, indicating that NF-kappaB is essential in p53-mediated cell death. Activation of NF-kappaB by p53 was distinct from that mediated by tumour-necrosis factor-alpha and involved MEK1 and the activation of pp90rsk. Inhibition of MEK1 blocked activation of NF-kappaB by p53 and completely abrogated p53-induced cell death. We conclude that inhibition of NF-kappaB in tumours that retain wild-type p53 may diminish, rather than augment, a therapeutic response.

Similarities and differences between human and murine TNF promoters in their response to lipopolysaccharide.

Transcription of the TNF gene is rapidly and transiently induced by LPS in cells of monocyte/macrophage lineage. Previous data suggested that multiple NF-kappaB/Rel binding sites play a role in the transcriptional response to LPS of the murine gene. However, the relevance of homologous sites in the human TNF gene remained a matter of controversy, partly because the high affinity NF-kappaB/Rel site located at -510 in the murine promoter is not conserved in humans. Here we used two sets of similarly designed human and mouse TNF promoter deletion constructs and overexpression of IkappaB in the murine macrophage cell line ANA-1 to show remarkable similarity in the pattern of the transcriptional response to LPS, further demonstrating the functional role of the distal promoter region located between -600 and -650. This region was characterized by mutagenesis of protein binding sites, including two relatively low affinity NF-kappaB/Rel sites, #2 and 2a. Mutation in each of the NF-kappaB sites resulted in 2- to 3-fold lower transcriptional activity in response to LPS. In contrast to LPS activation, the response to PMA was substantially lower in magnitude and required only the proximal promoter region. In summary, the functional topography of human and murine promoters when assayed in the same system has some marked similarities. Our observations support the notion that full LPS response of TNF gene requires both NF-kappaB and non-NF-kappaB nuclear proteins. Our data also suggest that the functional activity of a given kappaB site depends on the entire DNA sequence context in the promoter region.

E2F-1 potentiates cell death by blocking antiapoptotic signaling pathways.

The E2F family of transcription factors plays an essential role in promoting cell cycle progression, and one member of the family, E2F-1, is also capable of inducing apoptosis. We show here that E2F-1 can induce apoptosis by a death receptor-dependent mechanism, by downregulating TRAF2 protein levels and inhibiting activation of antiapoptotic signals including NF-kappa B. In this way, E2F-1 expression can lead to the sensitization of cells to apoptosis by a number of agents independently of p53. Deregulation of E2F-1 activity occurs in the majority of human tumors, and the ability of E2F-1 to inhibit antiapoptotic signaling may contribute to the enhanced sensitivity of transformed cells to chemotherapeutic agents.

The N-terminal domain of IkappaB alpha masks the nuclear localization signal(s) of p50 and c-Rel homodimers.

Members of the Rel/NF-kappaB family of transcription factors are related to each other over a region of about 300 amino acids called the Rel Homology Domain (RHD), which governs DNA binding, dimerization, and binding to inhibitor. At the C-terminal end of the RHD, each protein has a nuclear localization signal (NLS). The crystal structures of the p50 and RelA family members show that the RHD consists of two regions: an N-terminal section which contains some of the DNA contacts and a C-terminal section which contains the remaining DNA contacts and controls dimerization. In unstimulated cells, the homo- or heterodimeric Rel/NF-kappaB proteins are cytoplasmic by virtue of binding to an inhibitor protein (IkappaB) which somehow masks the NLS of each member of the dimer. The IkappaB proteins consist of an ankyrin-repeat-containing domain that is required for binding to dimers and N- and C-terminal domains that are dispensable for binding to most dimers. In this study, we examined the interaction between IkappaB alpha and Rel family homodimers by mutational analysis. We show that (i) the dimerization regions of p50, RelA, and c-Rel are sufficient for binding to IkappaB alpha, (ii) the NLSs of RelA and c-Rel are not required for binding to IkappaB alpha but do stabilize the interaction, (iii) the NLS of p50 is required for binding to IkappaB alpha, (iv) only certain residues within the p50 NLS are required for binding, and (v) in a p50-IkappaB alpha complex or a c-Rel-IkappaB alpha complex, the N terminus of IkappaB alpha either directly or indirectly masks one or both of the dimer NLSs.

Receptor-specific induction of NF-kappaB components in primary B cells.

The NF-kappaB transcription factor complex plays a key role in the expression of genes involved in immune responses. Nuclear NF-kappaB is induced in B lymphocytes by engagement of either the antigen receptor (sIg) or the CD40 receptor for a T cell activation antigen, although different intracellular pathways appear to be involved. In the present study the protein composition of NF-kappaB complexes triggered by sIg and CD40 was probed by electrophoretic mobility shift, supershift, shift-Western, and Western blot analyses. At the time of peak NF-kappaB induction (2 h), the NF-kappaB components detected in the complexes induced through sIg and through CD40 were the same. However, with continued stimulation RelB completely disappeared from anti-Ig-stimulated kappaB binding material, but remained a component of CD40L-induced NF-kappaB. The loss of DNA-binding RelB from anti-Ig-induced NF-kappaB did not result from depletion of RelB from B cell nuclei, suggesting specific regulation of RelB function which is not directly attributed to IkappaB function. These results indicate that NF-kappaB complexes may undergo protein-specific alterations in a time- and receptor-dependent fashion that may be associated with differences in the outcomes of B cell stimulation through sIg and CD40.

Interaction of NF-kappaB and NFAT with the interferon-gamma promoter.

Interferon-gamma (IFN-gamma) is a pleiotropic lymphokine whose production is restricted to activated T cells and NK cells. Along with other cytokines, IFN-gamma gene expression is inhibited by the immunosuppressant cyclosporin A. We have previously identified an intronic enhancer region (C3) of the IFN-gamma gene that binds the NF-kappaB protein c-Rel and that shows partial DNA sequence homology with the cyclosporin A-sensitive NFAT binding site and the 3'-half of the NF-kappaB consensus site. Sequence analysis of the IFN-gamma promoter revealed the presence of two additional C3-related elements (C3-1P and C3-3P). In addition, an NF-kappaB site (IFN-gamma kappaB) was identified within the promoter region. Based on this observation, we have analyzed the potential role of NF-kappaB and NFAT family members in regulating IFN-gamma transcription. Electrophoretic mobility shift assay analysis demonstrated that after T cell activation, the p50 and p65 NF-kappaB subunits bind specifically to the newly identified IFN-gamma kappaB and C3-related sites. In addition, we identified the NFAT proteins as a component of the inducible complexes that bind to the C3-3P site. Site-directed mutagenesis and transfection studies demonstrate that calcineurin-inducible transcriptional factors enhance the transcriptional activity of the IFN-gamma promoter through the cyclosporin-sensitive C3-3P site, whereas NF-kappaB proteins functionally interact with the C3-related sites. In addition, when located downstream to the beta-galactosidase gene driven by the IFN-gamma promoter, the intronic C3 site worked in concert with both the IFN-gamma kappaB and the C3-3P site to enhance gene transcription. These results demonstrate that the coordinate activities of NFAT and NF-kappaB proteins are involved in the molecular mechanisms controlling IFN-gamma gene transcription.

I kappa B epsilon, a novel member of the I kappa B family, controls RelA and cRel NF-kappa B activity.

We have isolated a human cDNA which encodes a novel I kappa B family member using a yeast two-hybrid screen for proteins able to interact with the p52 subunit of the transcription factor NF-kappa B. The protein is found in many cell types and its expression is up-regulated following NF-kappa B activation and during myelopoiesis. Consistent with its proposed role as an I kappa B molecule, I kappa B-epsilon is able to inhibit NF-kappa B-directed transactivation via cytoplasmic retention of rel proteins. I kappa B-epsilon translation initiates from an internal ATG codon to give rise to a protein of 45 kDa, which exists as multiple phosphorylated isoforms in resting cells. Unlike the other inhibitors, it is found almost exclusively in complexes containing RelA and/or cRel. Upon activation, I kappa B-epsilon protein is degraded with slow kinetics by a proteasome-dependent mechanism. Similarly to I kappa B-alpha and I kappa B, I kappa B-epsilon contains multiple ankyrin repeats and two conserved serines which are necessary for signal-induced degradation of the molecule. A unique lysine residue located N-terminal of the serines appears to be not strictly required for degradation. Unlike I kappa B- alpha and I kappa B-beta, I kappa B-epsilon does not contain a C-terminal PEST-like sequence. I kappa B-epsilon would, therefore, appear to regulate a late, transient activation of a subset of genes, regulated by RelA/cRel NF-kappa B complexes, distinct from those regulated by other I kappa B proteins.

IkappaBalpha deficiency results in a sustained NF-kappaB response and severe widespread dermatitis in mice.

The ubiquitous transcription factor NF-kappaB is an essential component in signal transduction pathways, in inflammation, and in the immune response. NF-kappaB is maintained in an inactive state in the cytoplasm by protein-protein interaction with IkappaBalpha. Upon stimulation, rapid degradation of IkappaBalpha allows nuclear translocation of NF-kappaB. To study the importance of IkappaBalpha in signal transduction, IkappaBalpha-deficient mice were derived by gene targeting. Cultured fibroblasts derived from IkappaBalpha-deficient embryos exhibit levels of NF-kappaB1, NF-kappaB2, RelA, c-Rel, and IkappaBbeta similar to those of wild-type fibroblasts. A failure to increase nuclear levels of NF-kappaB indicates that cytoplasmic retention of NF-kappaB may be compensated for by other IkappaB proteins. Treatment of wild-type cells with tumor necrosis factor alpha (TNF-alpha) resulted in rapid, transient nuclear localization of NF-kappaB. IkappaBalpha-deficient fibroblasts are also TNF-alpha responsive, but nuclear localization of NF-kappaB is prolonged, thus demonstrating that a major irreplaceable function Of IkappaBalpha is termination of the NF-kappaB response. Consistent with these observations, and with IkappaBalpha and NF-kappaB's role in regulating inflammatory and immune responses, is the normal development Of IkappaBalpha-deficient mice. However, growth ceases 3 days after birth and death usually occurs at 7 to 10 days of age. An increased percentage of monocytes/macrophages was detected in spleen cells taken from 5-, 7-, and 9-day-old pups. Death is accompanied by severe widespread dermatitis and increased levels of TNF-alpha mRNA in the skin.

Regulation of NF-kappa B activation in T helper 1 and T helper 2 cells.

In most cell types, NF-kappa B is activated by release from a cytoplasmic inhibitor protein, I kappa B, followed by its translocation to the nucleus where it binds to the regulatory regions of many genes, including the IL-2 gene in T lymphocytes. We have previously shown by electrophoretic mobility shift assays that nuclear extracts prepared from activated, non-IL-2-producing Th2 cell clones. We show here that Th-1 and Th2 cells have similar levels of cytoplasmic p65(RelA) and p50, but TCR stimulation fails to induce the nuclear translocation of p65(RelA) in Th2 cells. Nuclear translocation of p65(RelA) can be induced by IL-1 stimulation of Th2 cells, indicating that a basic mechanism of NF-Kappa B activation common to many cells is intact in Th2 cells. We demonstrate that IL-1 and TNF induce rapid nuclear translocation of p65(RelA) in T cell clones, whereas TCR-induced NF-Kappa B activation in Th1 cells is delayed and may be longer in duration. This suggests that the TCR pathway of NF-Kappa B activation is different from the cytokine pathway. Furthermore, we show that Th1 and Th2 cells express different levels and/or different forms of I kappa B alpha, and that cytokines, but not TCR stimuli, significantly modulate detectable levels of cytoplasmic I kappa B alpha.

Identification of an I kappa B alpha-associated protein kinase in a human monocytic cell line and determination of its phosphorylation sites on I kappa B alpha.

Nuclear factor kappa B (NF-kappa B) is stored in the cytoplasm as an inactive form through interaction with I kappa B. Stimulation of cells leads to a rapid phosphorylation of I kappa B alpha, which is presumed to be important for the subsequent degradation. We have recently reported the establishment of a lipopolysaccharide (LPS)-dependent cell-free activation system of NF-kappa B in association with the induction of I kappa B alpha phosphorylation. In this study, we have identified a kinase in cell extracts from the LPS-stimulated human monocytic cell line, THP-1, that specifically binds and phosphorylates I kappa B alpha. LPS stimulation transiently enhanced the I kappa B alpha-bound kinase activity in THP-1 cells. Mutational analyses of I kappa B alpha and competition experiments with the synthetic peptides identified major phosphorylation sites by the bound kinase as Ser and Thr residues in the C-terminal acidic domain of I kappa B alpha. Moreover, we show that the peptide, corresponding to the C-terminal acidic domain of I kappa B alpha, blocked the LPS-induced NF-kappa B activation as well as inducible phosphorylation of endogenous I kappa B alpha in a cell-free system using THP-1 cells. These results suggested that the bound kinase is involved in the signaling pathway of LPS by inducing the phosphorylation of the C-terminal region of I kappa B alpha and subsequent dissociation of the NF-kappa B.I kappa B alpha complex.

N- and C-terminal sequences control degradation of MAD3/I kappa B alpha in response to inducers of NF-kappa B activity.

The proteolytic degradation of the inhibitory protein MAD3/I kappa B alpha in response to extracellular stimulation is a prerequisite step in the activation of the transcription factor NF-kappa B. Analysis of the expression of human I kappa B alpha protein in stable transfectants of mouse 70Z/3 cells shows that, as for the endogenous murine protein, exogenous I kappa B alpha is degraded in response to inducers of NF-kappa B activity, such as phorbol myristate acetate or lipopolysaccharide. In addition, pretreatment of the cells with the proteasome inhibitor N-Ac-Leu-Leu-norleucinal inhibits this ligand-induced degradation and, in agreement with previous studies, stabilizes a hyperphosphorylated form of the human I kappa B alpha protein. By expressing mutant forms of the human protein in this cell line, we have been able to delineate the sequences responsible for both the ligand-induced phosphorylation and the degradation of I kappa B alpha. Our results show that deletion of the C terminus of the I kappa B alpha molecule up to amino acid 279 abolishes constitutive but not ligand-inducible phosphorylation and inhibits ligand-inducible degradation. Further analysis reveals that the inducible phosphorylation of I kappa B alpha maps to two serines in the N terminus of the protein (residues 32 and 36) and that the mutation of either residue is sufficient to abolish ligand-induced degradation, whereas both residues must be mutated to abolish inducible phosphorylation of the protein. We propose that treatment of 70Z/3 cells with either phorbol myristate acetate or lipopolysaccharide induces a kinase activity which phosphorylates serines 32 and that these phosphorylations target the protein for rapid proteolytic degradation, possibly by the ubiquitin-26S proteasome pathway, thus allowing NF-kappa B to translocate to the nucleus and to activate gene expression.

Conserved kappa B element located downstream of the tumor necrosis factor alpha gene: distinct NF-kappa B binding pattern and enhancer activity in LPS activated murine macrophages.

Transcriptional activation of various genes by lipopolysaccharide (LPS) is known to be mediated, at least in part, by the NF-kappa B/Rel family of transcription factors. We have identified a novel kappa B element located immediately downstream of the TNF-alpha gene that is conserved together with its flanking sequences across species lines and can act as an LPS-responsive enhancer for reporter gene constructs driven by the minimal TNF promoter. In extracts from activated murine macrophages and macrophage cell lines this element binds several non-canonical NF-kappa B/Rel complexes, in addition to p50 (NFKB1) homodimer and p50-p65 (NKFB1-RelA) heterodimer. Combination of high-resolution electrophoretic mobility shift assays (EMSA) with monospecific antibodies and u.v.-cross-linking indicates that the prominent slow migrating complex III contain p65 homodimer and c-Rel. The appearance of complex III in EMSA parallels the translocation of p65 and c-Rel into the nucleus and occurs shortly after LPS induction. Transfection experiments with reporter constructs driven by this kappa B element indicate strong inducibility by LPS and p65, moderate inducibility by c-Rel and repression by p50. Functional activity of sandwich TNF-CAT-TNF constructs further suggests that LPS-inducible transcriptional activation of the TNF gene in murine macrophages may be partly mediated by a downstream enhancer.