Pulmonary vein isolation for atrial fibrillation: Does ablation technique influence outcome?

BACKGROUND: Over the last 20 years various techniques have been developed striving for safer and more durable pulmonary vein isolation (PVI). The three most commonly used tools are pulmonary vein ablation catheter (PVAC) and cryoballoon ('single-shot' techniques), and point-by-point (PBP) radiofrequency ablation using 3D electroanatomical mapping (EAM). OBJECTIVE: Evaluate the safety and efficacy of the different techniques in an unselected population undergoing de-novo ablation for persistent or paroxysmal atrial fibrillation (AF) at Royal Papworth Hospital (RPH). METHOD: Retrospective, single-centre study of consecutive AF ablations at RPH between March 2017 and April 2018. Demographic, procedural and outcome data were analysed. RESULTS: Over the study period 329 first-time PVI procedures were performed. 37.4% were performed using PBP, 39.8% using cryoballoon and 22.8% using PVAC. There was no significant difference in age or sex between different ablation technique groups. 238 procedures were performed for paroxysmal AF and 91 for persistent AF. A higher proportion of the persistent cases were performed using point-by-point techniques compared to paroxysmal cases (58.2% vs 29.0%, p < 0.05). Procedural times were significantly longer in the group undergoing PBP ablation compared to cryoballoon or PVAC. However, there was no statistically significant difference in 12-month freedom from symptomatic AF or procedural complications between the groups. CONCLUSIONS: PBP, PVAC and cryoballoon AF ablation all appeared equally efficacious in an unselected population, though PVAC and cryoballoon procedures were shorter. All procedures were associated with a low adverse event rate. Prospective examination is required to substantiate this finding.

Heterocyclic homoprostanoid derivative attenuates monoarthritis in rats: An in vitro and in vivo preclinical paradigm.

From our lab, among the nineteen heterocyclic homoprostanoids (HHPs), three derivatives (compounds 3, 3b and 3c) exerted antioxidant and anti-inflammatory activity. Present study is an extension of the earlier work, and, is designed to establish their therapeutic potential in monoarthritis in rats. In addition, their possible mechanism of action would be investigated. A battery of in vitro tests such as lipopolysaccharide (LPS)-induced nitrite (NO)/reactive oxygen species (ROS) and NO/interleukin (IL)-6 generation in murine macrophages and whole blood (WhB), respectively were conducted. Later, in vitro cyclooxygenase (COX) enzyme inhibitory activity was also evaluated. All the tested compounds showed comparable efficacy against ROS and NO in LPS-stimulated murine macrophages. However, compound 3 did not exert inhibitory effect on LPS-induced NO/IL-6 generation in WhB assay. Compounds (3b and 3c) inhibited the NO generation in LPS-stimulated WhB. However, only compound 3b reversed the raised IL-6 levels in this assay. None of the test compounds inhibited COX iso-enzymes in the in vitro assay. All three HHPs showed comparable efficacy against carrageenan-induced paw inflammation. However, none of them exhibited any dose-dependent effect in this model. Based upon previous reports, compound 3c was explored against adjuvant-induced monoarthritis (AIA) in male Sprague-Dawley rats, where it exerted promising therapeutic effect. In addition to radiological and histological examinations of tibio-tarsal joint, various parameters such as chronic inflammation/pain, clinical score, interleukin (IL)-6 levels and complete blood cell profile were evaluated in AIA rats. Chronic treatment with 3c halted the disease progression in rats, improved the overall health of animals, as demonstrated by haematological, clinical scoring and joint examinations (radiological and histopathological). Inhibitory effect on elevated IL-6 in AIA rats suggested the possible mechanism of 3c on cytokine signalling. Overall, the study supports the anti-arthritic potential of compound 3c.

Novel 2,5-disubstituted-1,3,4-oxadiazoles as anti-inflammatory drugs.

OBJECTIVE: 1,3,4-oxadiazole ring is a versatile moiety with a wide range of pharmacological properties. The present work deals with the synthesis and evaluation of the anti-inflammatory activity of two novel 2,5-disubstituted-1,3,4-oxadiazoles (OSD and OPD). MATERIALS AND METHODS: Carrageenan-induced rat hind paw edema was employed as an acute model of inflammation. For evaluating sub-acute anti-inflammatory activity, carrageenan-induced inflammation in rat air pouch was employed. Complete Freund's adjuvant-induced arthritis in rats was used as a model of chronic inflammation. To evaluate in vitro anti-inflammatory activity, lipopolysaccharide (LPS)-stimulated RAW264.7 cells were used. RESULTS: OSD (100 mg/kg) reduced carrageen-induced paw edema by 60%, and OPD (100 mg/kg) produced a modest 32.5% reduction. OSD also reduced leukocyte influx and myeloperoxidase in carrageenan-induced rat air pouch model. In complete Freund's adjuvant-induced arthritis model, both OSD and OPD (200 mg/kg for 14 days) reduced paw edema and NO levels. In LPS-stimulated RAW264.7 cells, OSD and OPD inhibited formation of nitric oxide and reactive oxygen species, with OPD showing a better activity in comparison to OSD. CONCLUSIONS: OSD was the better of the two compounds in in vivo models of inflammation. The o-phenol substitution at position 2 of oxadiazole ring in OSD may be responsible for its better in vivo anti-inflammatory activity. The ability of the compounds to inhibit LPS-induced pro-inflammatory mediator release suggests an anti-inflammatory mechanism targeting LPS-TLR4-NF-κB signalling pathway, which needs to be explored in detail. The disparate efficacy in vitro and in vivo also requires in-depth evaluation of the pharmacokinetics of these novel oxadiazoles.

Atenolol and amlodipine combination overdose managed with continuous venovenous hemodiafiltration: A case report.

We present a patient of who ingested large dose of of atenolol and amlodipine and was treated successfully with continuous venovenous hemodiafiltration. Early recognition of indications for renal support and early initiation of the same is the key to successful management.

Antioxidant, anti-inflammatory and anti-hyperglycaemic activities of heterocyclic homoprostanoid derivatives.

A series of 19 heterocyclic homoprostanoids were synthesized from easily available oleic and ricinoleic acids and evaluated for their possible antioxidant, anti-inflammatory and anti-hyperlipidaemic activities. Compounds with thioxo- and oxoimidazole ring (1) and (2) have shown potent antioxidant activity with IC(50) values 0.23±0.09 and 0.41±0.01mM comparable with standard ascorbic acid. Compound (3) with a quinoxaline ring showed maximum inhibition of BSA denaturation at 1mM concentration and comparable with standard diclofenac. Incorporation of electron withdrawing substitutions like chloro- and nitro-groups in the quinoxaline ring has resulted in an increase anti-inflammatory activity. Test compounds (3), (3a) and (3c) showed modest inhibition of DPP-IV in vitro. However, the unsubstituted quinoxaline (3) and substituted quinoxalines (3b and 3c) reduced plasma glucose levels indicating the presence of hypoglycemic activity.

Synthesis and antitumor activity of optically active thiourea and their 2-aminobenzothiazole derivatives: a novel class of anticancer agents.

A novel series of optically active 2-aminobenzothiazole derivatives were synthesized by reaction of optically active amine (I) with thiophosgene to obtain optically active isothiocyanates (IIa-h) which on condensation with 4-fluoro-3-chloro aniline (III) yielded various optically active thioureas (IVa-h). Further oxidative cyclisation in the presence of bromine and chloroform yielded title compounds (Va-h). The structures of these compounds were established by IR, (1)H NMR, (13)C NMR, Mass and HRMS. The compounds (IVa-h and Va-h) were evaluated for in vitro cytotoxicity against mouse Ehrlich Ascites Carcinoma (EAC) and two human cancer cell lines (MCF-7 and HeLa). In preliminary MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cytotoxicity studies the optically active thiourea derivatives (IVe, IVf and IVh) were found most effective. In EAC cells the IC(50) values for IVe, IVf, IVh and Vg were found in the range of 10-24 microM, whereas in MCF-7 and HeLa cells the IC(50) values were observed in the range of 15-30 microM and 33-48 microM, respectively. In alkaline comet assay the compounds (IVe and IVf) showed dose-dependent DNA damaging activity.

Hdm2 is regulated by K-Ras and mediates p53-independent functions in pancreatic cancer cells.

There is emerging evidence that the oncogenic potential of hdm2 (human and/or murine double minute-2 protein) stems not only from its ability to counteract tumor suppressor p53 but also from its less understood p53-independent functions. Surprisingly, little is known about the role and regulation of hdm2 in pancreatic tumors, a large proportion (50-75%) of which contain mutant p53. In this study, we determined that hdm2 was expressed in a Ras-signaling-dependent manner in various pancreatic cancer cell lines. As p53 was mutated and inactive in these cells, the expression of hdm2 was seemingly redundant. Indeed, the proliferation and survival of cell lines such as Panc-1 and Panc-28 could be inhibited by PRIMA-1 (mutant p53 activator) but not by Nutlin-3 (inhibitor of the hdm2-p53 interaction). Unexpectedly, however, the proliferation of both cell lines was strongly inhibited by hdm2-specific RNAi. Our data also revealed cyclin D1, c-Jun and c-Myc to be novel targets of hdm2 and suggested that they might mediate hdm2's role in cellular proliferation and/or survival. We conclude from our results that hdm2 is expressed in pancreatic cancer cells as a result of activated Ras signaling, and that it regulates cellular proliferation and the expression of three novel target genes by p53-independent mechanisms.

c-Jun promotes cellular survival by suppression of PTEN.

Activation of c-Jun, a component of the AP-1 family of transcription factors, leads to either promotion or prevention of apoptosis. However, the molecular determinants of c-Jun-mediated cell survival are still unclear. We show here that inducible expression of c-Jun promotes cellular survival by negatively regulating the expression of the tumor-suppressor PTEN, resulting in the concomitant activation of the Akt survival pathway. Consistently, c-jun-/- fibroblasts, which are sensitive to nutrient deprivation, and human cell lines in which c-Jun expression is silenced, express elevated levels of PTEN. siRNA-mediated silencing of PTEN resulted in the reduction of cell-death owing to c-Jun deficiency. c-Jun was found to suppress PTEN expression by binding to a variant AP-1 site found in the 5' upstream sequences of PTEN promoter. Finally, an inverse correlation between c-Jun and PTEN levels was apparent in a panel of human tumor cell lines, independent of their p53 status. Together, the data demonstrate that c-Jun contributes to the promotion of cellular survival by regulating the expression of PTEN.

Antitumor activity of total alkaloid fraction of solanum pseudocapsicum leaves.

The total alkaloid fraction of the methanolic extract of Solanum pseudocapsicum leaves was tested for its in-vivo antitumor activity against Dalton's Lymphoma Ascites model in mice. The total alkaloid fraction at 2.5 and 5.0 mg/kg body weight doses exhibited antitumor activity as revealed by the significant increase in the mean survival time and the percentage increase in life span of tumor bearing mice. The antitumor activity observed may be due to its cytotoxic properties. However the treatment caused a significant decrease in the body weight below the normal indicating the toxicity of the treatment.

T cell antigen receptor vaccines for active therapy of T cell malignancies.

T cell lymphoproliferative disorders continue to be serious management problems, and so alternative therapeutic modalities are continuously being explored. One such strategy involves immunotherapy using the T cell receptor (TCR) as a target. Specifically we are attempting to develop a T cell receptor idiotype (TCR-Id) vaccine because the TCR-Id can serve as a tumor-specific antigen. In this article we will briefly review the rationale for TCR-Id vaccines, the preclinical models as developed in our laboratory, and a discussion of our current plans for a vaccine trial in mycosis fungoides.

Signaling pathways in pancreatic cancer.

Pancreatic cancer is, indisputably, one of the most malignant gastrointestinal tumors. Although the etiology of this disease is unknown, it is clearly linked to alterations in the biologic activities of various signaling molecules. Aberrant signaling activities of growth factors and their receptors, transcription factors, and proteins that control the cell cycle have been increasingly implicated in the pathogenesis and dissemination of pancreatic tumors. It is indeed possible that several of these molecules are, in fact, part of a signaling network that has gone awry. This review summarizes some recent advances in an attempt to generate a working model for future investigations.

Tumor suppressor MMAC/PTEN inhibits cytokine-induced NFkappaB activation without interfering with the IkappaB degradation pathway.

The phosphoinositide 3-kinase (PI 3-kinase) pathway has been implicated in the activation of the proinflammatory transcription factor nuclear factor kappaB (NFkappaB). To investigate the role of this pathway in NFkappaB activation, we employed mutated in multiple advanced cancers/phosphatase and tensin homologue (MMAC/PTEN), a natural antagonist of PI 3-kinase activity. Our results show that cytokine-induced DNA binding and transcriptional activities of NFkappaB were both inhibited in a glioma cell line that was stably transfected with MMAC/PTEN. The ability of interleukin-1 (IL-1) to induce inhibitor (IkappaB) degradation or nuclear translocation of NFkappaB was, however, unaffected by MMAC/PTEN expression, suggesting that PI 3-kinase utilizes another equally important mechanism to control NFkappaB activation. It is conceivable that NFkappaB is directly phosphorylated through such a mechanism because treatment with protein phosphatase 2A significantly reduced its DNA binding activity. Moreover, IL-1-induced phosphorylation of p50 NFkappaB was potently inhibited in MMAC/PTEN-expressing cells. Whereas the mediators of NFkappaB phosphorylation remain to be identified, IL-1 was found to induce physical interactions between the PI 3-kinase target Akt kinase and the IkappaB.IkappaB kinase complex. Physical interactions between these proteins were antagonized by MMAC/PTEN consistent with their potential involvement in NFkappaB activation. Taken together, our observations suggest that PI 3-kinase regulates NFkappaB activation through a novel phosphorylation-dependent mechanism.

Favorable treatment outcome in non-Hodgkin's lymphoma patients with "poor" mobilization of peripheral blood progenitor cells.

Our purpose was to evaluate the outcome and costs of high-dose chemotherapy and autologous peripheral blood progenitor cell (PBPC) transplantation in patients with the inability to mobilize sufficient numbers of PBPCs to allow rapid engraftment after PBPC transplantation. We treated 172 consecutive non-Hodgkin's lymphoma (NHL) patients with cyclophosphamide and granulocyte colony-stimulating factor followed by apheresis to collect PBPCs. The cells were separated on a Percoll gradient and purged with monoclonal antibodies and complement. The patients were categorized as "good" mobilizers if a collection of > or =2 x 10(6) CD34+ cells/kg was obtained (n = 138, 80%) or "poor" mobilizers if <2 x 10(6) CD34+ cells/kg were obtained (n = 34, 20%). With a median follow-up of 3.5 years, there is no statistically significant difference in actuarial event-free survival, overall survival, or relapse for good mobilizers compared with poor mobilizers. However, there was a trend toward increasing nonrelapse, transplantation-related mortality of 11.8% for poor mobilizers versus 3.6% for good mobilizers (P = .08) and early death from all causes including relapse within 120 days (poor 20.6% versus good 8.7%, P = .06). The total cost for bone marrow transplantation-related care was significantly higher, at $140,264 for poor mobilizers versus $80,833 for good mobilizers (P = .0001). The population of patients with NHL who mobilize PBPCs poorly into the circulation have a higher cost for posttransplant support. However, there is no significant difference in relapse, event-free survival, or overall survival for such patients compared with those who mobilize PBPCs easily.

Sensitivity of myelomonocytic leukemia cells to arsenite-induced cell cycle disruption, apoptosis, and enhanced differentiation is dependent on the inter-relationship between arsenic concentration, duration of treatment, and cell cycle phase.

Arsenite treatment has been found to induce clinical remission in patients with acute promyelocytic leukemia. Although the potential therapeutic value of arsenite may lie in triggering apoptosis, it has not been established that cytotoxicity is the sole mechanism of action. We have used a myelomonocytic leukemia cell line (U937) to characterize the concentration-dependent effects of arsenite on cell growth, viability, apoptosis, and differentiation. Arsenite has multiple effects on U937 cells. Low concentrations of arsenite (i.e., < or = 1 microM) potentiate vitamin-D(3)-induced differentiation. Two markers of monocyte differentiation, Mac-1 expression and nitroblue tetrazolium reduction, are increased in arsenite-exposed, D(3)-costimulated cells. Concentrations of arsenite >10 microM rapidly induce the death of cells irrespective of cell cycle phase. Intermediate concentrations of arsenite (i.e., 5 to 10 microM) are cytostatic initially. Cell cycle analysis using elutriated, synchronous cell populations revealed that intermediate concentrations of arsenite delay both G(1) and G(2) transit. G(2) cells appear to be most sensitive to arsenite, in that transit through G(2)/M is more delayed than transit through G(1), and apoptosis is induced in these cells as they emerge from an aberrant G(2)/M. Arsenite-induced apoptosis was caspase-3 dependent. Arsenite-mediated cytotoxicity was reduced in the presence of the broad caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone; however, caspase inhibition did not reverse arsenite-induced cytostasis. Thus, arsenite has multiple effects on U937 cells that are dependent on concentration and cell cycle phase. Specifically, cell cycle transit and differentiation are more sensitive to arsenite than is the induction of apoptosis.

Phosphatidylinositol 3-kinase as a mediator of TNF-induced NF-kappa B activation.

The activation of transcription factor NF-kappa B by TNF involves the stimulation of a novel signaling cascade. In this paper we show that phosphatidylinositol 3-kinase (PI 3-kinase) may play a pivotal role in TNF-mediated activation of NF-kappa B-dependent genes. Consistent with its involvement in TNF signaling, PI 3-kinase activities in HepG2 and U937 cells can be stimulated by TNF in a rapid but transient manner through a mechanism that may involve its association with the insulin receptor substrate-1. A dominant-negative mutant of the p85 regulatory subunit of PI 3-kinase, which is a potent inhibitor of PI 3-kinase signaling, effectively blocked the TNF-induced expression of an NF-kappa B-dependent reporter gene. Although PI 3-kinase may be required for NF-kappa B activation, overexpression of its p110 catalytic subunit alone was unable to induce an NF-kappa B/chloramphenicol acetyltransferase (CAT) reporter gene. However, when TNF was added to p110-overexpressing cells, there was a synergistic activation of the NF-kappa B/CAT reporter, suggesting that other TNF-inducible signals may cooperate with PI 3-kinase to activate NF-kappa B. Consistent with its role in NF-kappa B activation, inhibition of PI 3-kinase activity by wortmannin or LY294002 greatly potentiated TNF-induced apoptosis. This TNF/wortmannin-induced apoptosis was markedly prevented in cells overexpressing Rel A. Taken together, our results indicate that a PI 3-kinase-regulated step in TNF-signaling is critical for the expression of NF-kappa B-dependent genes.

Purification and identification of a tissue-specific repressor involved in serum amyloid A1 gene expression.

We have previously demonstrated that the 5'-flanking regions from the rat serum amyloid A1 (SAA1) promoter are necessary and sufficient to confer specific cytokine-induced expression in cultured hepatoma cells. Deletion analysis identified a tissue-specific repressor (TSR) regulatory element, located between bp -289 and -256, that functioned as a silencer, contributing to the transcription repression on SAA1 promoter in nonliver cells. When this 34-base pair TSR-binding element was used as a probe in electrophoretic mobility shift assays, an intense DNA-protein complex was detected in nuclear extracts from HeLa and several other nonliver tissues. This TSR binding activity, however, was undetectable in extracts from liver or liver-derived cells. The distribution of TSR binding activity is therefore consistent with its regulatory role in repressing SAA1 expression in a tissue-specific manner. In this study, we purified TSR protein from HeLa nuclear extracts and showed that it has a molecular mass of approximately 50 kDa. Surprisingly, protein sequencing and antibody supershift experiments identified TSR as transcription factor AP-2. Subsequent functional analysis showed that forced expression of AP-2 in HepG2 cells could indeed inhibit conditioned medium-induced SAA1 promoter activation. Moreover, expression of a dominant-negative mutant of AP-2 in HeLa cells or mutation of the AP-2-binding site led to derepression of the SAA1 promoter, presumably by neutralizing the inhibitory effects of the endogenous wild-type AP-2. Our results therefore demonstrate a novel function for AP-2 in the transcriptional repression of SAA1 promoter. Together with its tissue distribution, AP-2 may contribute to SAA1's highly liver-specific expression pattern by restricting its expression in nonliver cells.

Purification and characterization of the serum amyloid A3 enhancer factor.

Serum amyloid A (SAA) is a major acute-phase protein synthesized and secreted mainly by the liver. In response to acute inflammation, its expression may be induced up to 1000-fold, primarily as a result of a 200-fold increase in the rate of SAA gene transcription. We showed previously that cytokine-induced transcription of the SAA3 gene promoter requires a transcriptional enhancer that contains three functional elements: two CCAAT/enhancer-binding protein (C/EBP)-binding sites and a third site that interacts with a constitutively expressed transcription factor, SAA3 enhancer factor (SEF). Each of these binding sites as well as cooperation among their binding factors is necessary for maximum transcription activation by inflammatory cytokines. Deletion or site-specific mutations in the SEF-binding site drastically reduced SAA3 promoter activity, strongly suggesting that SEF is important in SAA3 promoter function. To further elucidate its role in the regulation of the SAA3 gene, we purified SEF from HeLa nuclear extracts to near homogeneity by using conventional liquid chromatography and DNA affinity chromatography. Ultraviolet cross-linking and Southwestern experiments indicated that SEF consisted of a single polypeptide with an apparent molecular mass of 65 kDa. Protein sequencing and antibody supershift experiments identified SEF as transcription factor LBP-1c/CP2/LSF. Cotransfection of SEF expression vector with SAA3-luciferase reporter resulted in approximately a 5-fold increase in luciferase activity. Interestingly, interleukin-1 treatment of SEF-transfected cells caused dramatic synergistic activation (31-fold) of the SAA3 promoter. In addition to its role in regulating SAA3 gene expression, we provide evidence that SEF could also bind in a sequence-specific manner to the promoters of the alpha(2)-macroglobulin and Aalpha-fibrinogen genes and to an intronic enhancer of the human Wilm's tumor 1 gene, suggesting a functional role in the regulation of these genes.

Apical extrusion of debris using two hand and two rotary instrumentation techniques.

The purpose of this study was to investigate the quantity of apical debris produced in vitro using two hand and two rotary instrumentation techniques. Sixty minimally curved, mature human mandibular premolars with single canals were divided into 4 groups of 15 teeth each and prepared using step-back instrumentation with K-files, balanced force with Flex-R files, Lightspeed nickel-titanium instruments, or .04 taper ProFile Series 29 rotary nickel-titanium files. Debris extruded through the apical foramen during instrumentation was collected on preweighed filters. The mean weight of extruded debris for each group was statistically analyzed using a Kruskal Wallis one-way analysis of variance and a Mann-Whitney U rank sum tested. Although all instrumentation techniques produced apically extruded debris, step-back instrumentation produced significantly more debris than the other methods (p < 0.0001). There was no difference between balanced force hand instrumentation and the two rotary nickel-titanium instrumentation methods (p > 0.05). Hand or engine-driven instrumentation that uses rotation seems to reduce significantly the amount of debris extruded apically when compared with a push-pull (filing) technique. Decreased apical extrusion of debris has strong implications for a decreased incidence of postoperative inflammation and pain.

Phosphatidylinositol 3-kinase in interleukin 1 signaling. Physical interaction with the interleukin 1 receptor and requirement in NFkappaB and AP-1 activation.

The signaling mechanisms utilized by the proinflammatory cytokine interleukin-1 (IL-1) to activate the transcription factors NFkappaB and activator protein-1 (AP-1) are poorly defined. We present evidence here that IL-1 not only stimulates a dramatic increase in phosphatidylinositol 3-kinase (PI 3-kinase) activity but also induces the physical interaction of its type I receptor with the p85 regulatory subunit of PI 3-kinase. Furthermore, two PI 3-kinase-specific inhibitors, wortmannin and a dominant-negative mutant of the p85 subunit, inhibited IL-1-induced activation of both NFkappaB and AP-1. Transient transfection experiments indicated that whereas overexpression of PI 3-kinase may be sufficient to induce AP-1 and increase nuclear c-Fos protein levels, PI 3-kinase may need to cooperate with other IL-1-inducible signals to fully activate NFkappaB-dependent gene expression. In this regard, cotransfection studies suggested that PI 3-kinase may functionally interact with the recently-identified IL-1-receptor-associated kinase to activate NFkappaB. Our results thus indicate that PI 3-kinase is a novel signal transducer in IL-1 signaling and that it may differentially mediate the activation of NFkappaB and AP-1.

Reconstitution of nuclear factor kappa B activation induced by tumor necrosis factor requires membrane-associated components. Comparison with pathway activated by ceramide.

Tumor necrosis factor (TNF) is known to induce the activation of a nuclear transcription factor, nuclear factor kappa B (NF-kappa B), in a wide variety of cell types. The post-receptor binding events that culminate in TNF-dependent NF-kappa B activation are not understood. To dissect this pathway, we developed a reconstitution system consisting of membrane, cytosolic, and post-nuclear fractions. Our results indicate that when incubated with the post-nuclear fraction derived from TNF-untreated cells, the membrane fraction from TNF-treated cells causes the activation of NF-kappa B with kinetics similar to that observed in intact cells. Under these conditions, the cytosolic fraction has no effect. This activation is tyrosine kinase-dependent since erbstatin completely abolished the effect. Furthermore, as revealed by immunoblotting, no degradation of the inhibitory subunit of NF-kappa B was observed. In this reconstitution system, we can also demonstrate the activation of NF-kappa B by ceramide, but this activation is not tyrosine kinase-dependent. Overall, our results indicate that intermediates required for NF-kappa B activation by TNF or ceramide are membrane-bound, but the mechanism of activation by TNF is most likely different from that of ceramide.