NF κB Research Articles

Synergistic interaction of proteasome and topoisomerase II inhibition in multiple myeloma

Multiple myeloma is a malignancy of terminally differentiated plasma cells and is incurable in the majority of the patients. Thus, novel effective treatment regimens are urgently needed. In this study, we examined the effects of co-treatment with proteasome-inhibitor bortezomib and topoisomerase II inhibitor etoposide in multiple myeloma cells lines OPM-2, RPMI-S and NCI-H929. Using the median effect method of Chou and Talalay, we evaluated the combination indices (CI) for simultaneous and sequential treatment schedules. In the sequential treatment schedule, we found strong synergistic effects in all three cell lines, even at low single-agent cytotoxicity levels. When cells were treated simultaneously with both drugs, the synergy was present but less pronounced than in the sequential treatment schedule. The synergistic effects observed in the co-treatment schedules were accompanied by an inhibition of anti-apoptotic effects that were induced by etoposide alone. Namely, bortezomib abrogated both etoposide-induced NF–κB activation and etoposide-induced bcl-2 up-regulation. Our data suggest that combining etoposide with bortezomib might be useful for cancer treatment, as bortezomib potentially inhibits counter-regulatory mechanisms of tumor cells, which are induced by topoisomerase II inhibition and which may contribute to acquired chemoresistance.

AGE–modified albumin containing infusion solutions boosts septicaemia and inflammation in experimental peritonitis

HSA preparations for i.v. use are administered in critically ill patients. Although increasing intravascular osmotic pressure seems to be a pathophysiologically orientated treatment, clinical trials do not indicate a benefit for mortality in HSA-treated patients. Instead, there is evidence for inflammatory reactions upon infusion of different HSA batches. A neglected issue concerning the safety and quality of these therapeutics is processing-related post-transcriptional protein modifications, such as AGEs. We therefore tested the hypothesis that commercially available infusion solutions contain AGEs and studied whether these protein modifications influence outcome and inflammation in a murine model of sepsis induced by CLP. Screening of different HSA and Ig preparations in this study revealed an up to approximate tenfold difference in the amount of AGE modifications. Application of clinically relevant concentrations of CML-modified HSA in CLP led to increased inflammation and enhanced mortality in wild-type mice but not in mice lacking the RAGE. Lethality was paralleled by increased activation of the proinflammatory transcription factor NF-kappaB, NF-kappaB-dependent gene expression, and infiltration of inflammatory cells in the peritoneal cavity. This study implies that infusion solutions containing a high load of the AGE-modified protein have the potential to activate RAGE/NF-kappaB-mediated inflammatory reactions, causing increased mortality in experimental peritonitis.

The Flavonoid, Quercetin, Inhibits HIV-1 Infection in Normal Peripheral Blood Mononuclear Cells

Problem statement: Flavonoids and related polyphenols, have been known to possess cardioprotective, anti-tumor, anti-inflammatory, an ti-allergic and anti-viral activities. Previous stu dies have shown that flavonoid, quercetin significantly downregulates expression of pro-inflammatory cytokines in cultured cells via modulation of NF κB and p38MAPK signaling pathways. In the current study, we hypothesize that quercetin exerts anti-HI V activity by differential modulation of pro-and anti-inflammatory cytokine expression in normal PBMCs. Approach: Cultures of PBMC received either different concentrations of quercetin (1-50 µM) or media alone. The anti-HIV effects of quercetin was evaluated in an in vitro infection model by quantitaing the HIV-1 LTR gene suppression by quantitative Real Time PCR, HIV-1 p24 antigen production by ELISA and viral infectivity by MAGI cell assay. Results: Our results showed that quercetin significantly do wnregulates p24 antigen production, LTR gene expression and viral infectivi ty in a dose dependent manner (5-50 μM) as compared to HIV infected untreated control PBMCs. Further, we reported that quercetin significantly downregulated the expression of the pro-inflammator y cytokine, TNF-α with concomitant upregulation of anti-inflammatory cytokine IL-13 as determined b y measurement of gene expression and protein production. A higher level of IL-13 is known to inh ibit TNF-α production and also HIV-1 infection. Thus, differential modulation of pro-and anti-infla mmatory cytokines could be one of the possible mechanisms for the anti-HIV effects of quercetin. Conclusion: Better understanding of the mechanisms underlying the anti-HIV effects of quercetin may help to develop a new neutraceutical agent useful in the treatment of HIV-1 infected sub jects in conjunction with conventional therapeutic regimens.

Linear polyubiquitylation: the missing link in NF–κB signalling

Metazoan cells respond to external stress or infection through multiple signalling pathways that converge on NF–κB activation, initiating a program designed to promote survival or trigger cell death. Recent evidence reveals a mechanism for NF-κB activation that requires assembly of linear polyubiquitin chains as scaffolds to recruit the activation complex.

The Role of the Microenvironment for CLL Proliferation and Survival: Gene Expression Profiling of Leukemic Cells Derived from Blood, Bone Marrow and Lymph Nodes Reveals the B-Cell Receptor and NF κB as Dominant Signaling Pathways

The host microenvironment is important for proliferation and survival of leukemic cells in chronic lymphocytic leukemia (CLL). Numerous molecules, signaling pathways and cell types have been reported to enhance CLL cell survival. To date, most reports on such interactions are derived from in-vitro studies, where each study focused on a specific ligand/receptor interaction or candidate pathway. Here, we adopted a more global approach to evaluate in-vivo effects of the microenvironment on leukemic cell biology. CLL cells from 15 patients were obtained on the same day from 3 different compartments: peripheral blood (PB), bone marrow (BM) and lymph node (LN), from which a single cell suspension was prepared. Tumor cells from all three compartments were purified by CD19 selection to purity >98%. Patients were assigned to prognostic subtypes based on immunoglobulin sequencing (Ig) and ZAP70 expression: 10 patients had the more progressive subtype (Ig-unmutated, ZAP70+) and 5 patients belonged to the more indolent subtype. Cells were analyzed for surface markers by flow cytometry and by gene expression profiling on Affymetrix HG U133 Plus 2.0 arrays. By flow cytometry, CLL cells in LN expressed higher levels of activation markers including CD69 and CD38 compared to CLL cells in PB (% CD19+/69+; 71 ±27 vs. 35 ±28, p<0.001 and % CD19+/CD38+; 33 ±28 vs. 20±19, p<0.001, respectively). The expression of activation markers in BM derived cells was less consistent and did not reach statistically significant differences. We therefore focused our analysis on a comparison between LN and PB derived cells. First, we confirmed that the expression of a diagnostic CLL gene expression signature established previously for PB derived cells (Klein et al, 2001) was equally present in leukemic cells derived from all three compartments. We then identified a set of about 275 genes that were differentially expressed between LN resident and circulating tumor cells, most of which were up-regulated (fold change >2, FDR <0.2). A large number of these genes encode proteins important for cell cycle control and proliferation: different cyclins, PCNA, Ki67, TOP2A and MYC. We also detected a significant increase in the expression of NF-κB target genes in LN resident tumor cells, including CD83, CD69, JunB, Cyclin D2, GADD45B, CCL3, CCL4 and others. Consistent with activation of the NF-κB pathway in LN, IκB-beta protein levels in tumor cells from LN were lower than levels in matching PB cells. Next we identified genes differentially expressed between CLL subtypes based on Ig-mutation status separately for each of the 3 compartments. Interestingly, these subtype identifying gene sets were only partially overlapping. In Ig-unmutated, ZAP70+ cells several genes were more strongly regulated by the microenvironment then in Ig-mutated, ZAP70 negative cells. Among these genes is LPL, which has been reported to distinguish the CLL subtypes, and other genes induced by B-cell receptor (BCR) signaling. Using in-vitro IgM activation, we show that these genes are indeed induced by BCR stimulation but not by CD40 ligation and that their induction is confined to ZAP70+ CLL cells. In conclusion: interactions between CLL cells and elements of the microenvironment in LN induce cell proliferation and NF-κB activation. The preferential upregulation of BCR regulated genes in ZAP70+ CLL demonstrates a more efficient in-vivo response of ZAP-70+ cells to BCR stimulation. Our results highlight the importance of NFκ κB and BCR signaling in CLL and provide a rationale to focus treatment approaches on these central pathways.

NF–κB activation in renal tubular epithelial cells by oxalate stimulation

The transcription factor nuclear factor-kappaB (NF-kappaB) is involved in inflammatory and immune responses through the induction of various cytokines and growth factors. Recently, the coordinated action of NF-kappaB and activator protein-1 was reported in osteopontin (OPN) expression. In the present study, we demonstrated that oxalate induces OPN expression by activating NF-kappaB in renal tubular cells. Furthermore, we investigated the inhibitory effect of N-acetyl-L-cysteine (NAC) on NF-kappaB activation in the human renal tubular cell line. All of the experiments were carried out using human kidney-2 cells, which are human proximal tubular epithelial cells immortalized by transduction with the human papillomavirus 16E6/E7 gene. The time-dependent extraction of total protein was performed after the uptake of 0.5 mM oxalate by the cells. The NF-kappaB activation and OPN expression were examined by western blotting and immunocytochemistry. As a result of oxalate stimulation, the amount of p65 subunit in the nucleus increased significantly (P < 0.05), and NAC significantly inhibited the translocation of p65 into the nucleus (P < 0.05). These observations indicate that NAC can be used as a drug to prevent stone formation.

Advanced Glycation End-Product of Low Density Lipoprotein Activates the Toll-Like 4 Receptor Pathway Implications for Diabetic Atherosclerosis

Diabetes is a major risk factor for coronary heart disease. Accumulation of advanced glycation end-products (AGEs) attributable to hyperglycemia in diabetics promotes the development of atherosclerosis. However, the underlying mechanisms remain unclear. The advanced glycation end-product of low-density-lipoprotein (AGE-LDL) induced proinflammatory cytokine production in human coronary artery endothelial cells and human- and mouse-macrophages. AGE-LDL stimulated cytokine synthesis was markedly reduced in mouse macrophages with a TLR4 loss-of-function mutation. Coimmunoprecipitation experiments indicated AGE-LDL interacts with TLR4, RAGE, and CD36. Incubation of cultured macrophages with TLR4, RAGE, or CD36 antibodies inhibited AGE-LDL stimulation of tumor necrosis factor (TNF)alpha production. A competitive binding inhibitor of TLR4 blocked AGE-LDL binding to the receptor. After transfection of a HEK293 cell system with wild-type TLR4, AGE-LDL activated a signaling pathway including p38 alpha, JNK, and ERK1 kinases and AP1, Elk1, and NF-kappaB transcription factors; the net result being increased cytokine production. These effects were absent when cells were transfected with empty plasmid. Two common polymorphisms in TLR4, D299G and T399I, reduced the response of TLR4 to lipopolysaccharide (LPS) but had no effect on AGE-LDL signaling. These results indicate that AGE-LDL activates a TLR4-mediated signaling pathway, thus inducing proinflammatory cytokine production. This mechanism may partly explain the increased risk of atherosclerosis observed in diabetics.

DMNQ-S17 inhibits constitutive NF-kappaB activation leading to induction of apoptosis through the activation of caspase-3 in human myeloid leukemia U937 cells

Through cytotoxicity screening with naphthoquinone derivatives, a novel compound 6-(1-oxoallkyl)-5,8-dimethoxy-1,4-naphthoquinone-S17 (DMNQ-S17) showed its potency against human myeloid leukemia U937 cells. Thus, to elucidate the apoptotic mechanism of DMNQ-S17, this study was performed in myeloid leukemia U937 cells by 2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) assay, eletrophoretic mobility shift assay (EMSA), terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay, 4′,6-diamidino-2-phenylindole (DAPI) staining, and Western blotting. In the present study, DMNQ-S17 inhibited constitutive NF κB activation and its transcriptional activity in U937 cells. In addition, DMNQ-S17 induced apoptotic features such as apoptotic bodies, cell shrinkage and chromatin condensation in U937 cells. Consistently, flow cytometric analysis showed that DMNQ-S17 increased sub-G1 portion and TUNEL positive cells in a concentration-dependent manner. Furthermore, DMNQ-S17 effectively attenuated mitochondrial membrane potential, released cytochrome C, activated caspase-3 expression, and cleaved poly (ADP-ribose) polymerase (PARP). Reversely, caspase-3 and -9 inhibitors also blocked the DMNQ-S17 induced caspase-3 activation and PARP cleavage in U937 cells. Taken together, these findings suggest that DMNQ-S17 can be a potent anticancer candidate for myeloid leukemias by the suppression of NF-κB activation leading to the activation of caspase-3 in human myeloid leukemia U937 cells.

P4-194: Presenilin-dependent gamma-secretase proteolysis of cytokine receptors

Following the identification of presenilins and their genetic association with the onset of Familial Alzheimer's disease, independent studies have shown that presenilins are the catalytic component of the gamma-secretase complex associated with the proteolytic cleavage of Amyloid Precursor Protein (APP), generation of amyloid-beta peptides and onset of Alzheimer's disease. Recently, many groups have revealed several additional presenilin-interacting proteins and gamma-secretase cleavage substrates, suggesting a diversity of roles for presenilin during development and adult life, some of which might contribute to Alzheimer's disease progression. Cell culture, Western blot, co-immunoprecipitation, molecular cloning. In our studies we have demonstrated that several members of the tumour necrosis factor (TNF)/nerve growth factor (NGF) and interleukin-1 (IL-1)/toll-like receptor (TLR) super families can be proteolytically processed in a manner similar to APP. We show that select members of the TNF/NGF and IL-1/TLR super families undergo ligand-dependant intramembrane proteolytic processing, mediated by presenilin-dependent γ-secretase activity. A metalloprotease-dependent proteolytic event liberates soluble receptor-ectodomains and produces membrane-associated C-terminal fragments (CTF). These membrane-anchored CTFs are substrates for subsequent γ-secretase cleavage, which generates TNF/NGF and IL-1/TLR derived intracellular domains (ICD). Generation of the ICD's is independently inhibited by specific pharmacological γ-secretase inhibitors, expression of dominant negative presenilin-1 or presenilin deficiency. Attenuation of γ-secretase activity or presenilin deficiency also impairs responsiveness to TNF/NGF and IL-1/TLR ligand-stimulated activation of the mitogen activated protein kinases (MAPK), nuclear factor κB (NF–κB) and cytokine secretion. Our data suggests that increased proteolytic processing and secretion of these cytokine and growth factor receptors may, through signalling deficits in the associated pathways, mediate nervous system inflammation in patients with Alzheimer's disease.

P3-371: Interleukin-1β enhances nucleotide-induced α-secretase–dependent amyloid precursor protein processing in rat primary cortical neurons via upregulation of the P2Y 2 receptor

The P2Y2 nucleotide receptor (P2Y2R) stimulates α-secretase-dependent cleavage of the amyloid precursor protein (APP) in human 1321N1 astrocytoma cells, causing extracellular release of the neuroprotective protein sAPPα. In this study, we analyzed functional upregulation of the P2Y2R by cytokines and the P2Y2 receptor-mediated production of sAPPα in rat primary cortical neurons (rPCNs). In rPCNs, P2Y2R mRNA and receptor activity were virtually absent in unstimulated cells, whereas overnight treatment with the inflammatory cytokine interleukin 1β (IL-1β) upregulated both P2Y2R mRNA expression and receptor activity. The upregulation of P2Y2R mRNA was abrogated by pre-incubation with an IkappaBα phosphorylation inhibitor Bay 11, which suggests that P2Y2R mRNA transcript levels are regulated through NF–κB signaling. Furthermore, the P2Y2R agonist UTP enhanced the release of sAPPα in rPCNs treated with IL-1β or transfected with P2Y2R mRNA. UTP-induced release of sAPPα from rPCNs was completely inhibited by pretreatment of the cells with the metalloproteinase inhibitor TAPI-2 or the phosphatidylinositol 3-kinase (PI3K) inhibitor LY2904, and was partially inhibited by the MEK inhibitor U0126 and unaffected by the protein kinase C (PKC) inhibitor GF109203. The data indicate that P2Y2R-mediated release of sAPPα from neurons is dependent on ADAM10/17 and PI3K activity, partially dependent on ERK1/2 activity, and does not require activation of PKC. These results suggest that under inflammatory conditions, IL-1β evokes the functional upregulation of the P2Y2R in neurons to promote ADAM10/17-dependent APP processing through P2Y2R-mediated activation of PI3K. Ongoing studies with the TgCRND8 mouse model of Alzheimer's Disease and P2Y2 knockout mice are evaluating these findings in vivo.

Biological response of human bone cells to zinc-modified Ca–Si-based ceramics

Calcium silicate (CaSiO 3) ceramics have received considerable attention in recent years due to their excellent bioactivity and degradability. However, their poor chemical stability limits their biological applications. Hardystonite (Ca 2ZnSi 2O 7) ceramics are Ca–Si-based materials developed by incorporating zinc into the Ca–Si system to improve their chemical stability. However, the biological responses of Ca 2ZnSi 2O 7 to bone cells are unknown. The objective of this study is to investigate and compare the in vitro responses of human osteoblast-like cells (HOBs) and osteoclasts when cultured on Ca 2ZnSi 2O 7 and CaSiO 3 ceramic disks. The ability of Ca 2ZnSi 2O 7 ceramics to support HOB attachment, cytoskeleton organization, proliferation and differentiation was assessed by scanning electron microscopy, confocal microscopy, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, alkaline phosphatase activity and quantitative real-time polymerase chain reaction. Our results show that Ca 2ZnSi 2O 7 supported HOB attachment with a well-organized cytoskeleton structure, and significantly increased cellular proliferation and differentiation compared to CaSiO 3. In addition, Ca 2ZnSi 2O 7 showed increased expression levels of osteoblast-related mRNAs (alkaline phosphatase, collagen type I, osteocalcin, receptor activator of NF κB ligand and osteoprotegerin) compared to CaSiO 3. Ca 2ZnSi 2O 7 ceramic supported the formation of mature and functional osteoclasts and formed resorption imprints. On CaSiO 3 ceramics, the cells failed to differentiate from the monocytes into osteoclasts. Taken together, these results indicate that Hardystonite ceramics are conducive to both types of bone cells, osteoblast-like cells and osteoclasts, suggesting their potential use for skeletal tissue regeneration and as coatings onto currently available orthopedic and dental implants.

Pouterin, a novel potential cytotoxic lectin-like protein with apoptosis-inducing activity in tumorigenic mammalian cells

In this study, the cytotoxicity of pouterin in tumorigenic and non-tumorigenic mammalian cell lines was investigated. We found that HeLa, Hep-2 and HT-29 tumor cells were highly sensitive to pouterin cytotoxicity in a dose-dependent manner, whereas non-tumorigenic Vero cells and human lymphocytes were relatively resistant to the protein. Among the tumor cell lines, HeLa cells showed the highest susceptibility to pouterin cytotoxicity, exhibiting a time-dependent increase in LDH leakage and an IC 50 value of 5 μg/mL. Morphological alterations such as rounding, cell shrinkage and chromatin condensation, consistent with apoptotic cell death were observed. Apoptosis induction was demonstrated by DNA fragmentation as detected by terminal dUTP nick-end labeling (TUNEL). Furthermore, HeLa cells incubated with pouterin showed disruption of the actin cytoskeleton. Western blot analysis revealed that pouterin caused increased expression of p21, thus indicating cell cycle arrest. Subsequent studies provided evidence that apoptosis may be partially explained in the activation of the tumor necrosis factor receptor 1 (TNFR1) signaling. Interestingly, a time-dependent decrease of the expression of p65 nuclear factor kappa B (NF κB) subunit, concomitant with a downregulation of the inhibitor of apoptosis protein 1 (IAP1) was observed, suggesting that TNFR-mediated apoptosis is the predominant pathway induced by pouterin in HeLa cells.

Asymmetric dimethylarginine induces TNF-α production via ROS/NF–κB dependent pathway in human monocytic cells and the inhibitory effect of reinioside C

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, has been implicated in vascular inflammation through induction of reactive oxygen species (ROS) and proinflammatory genes in endothelial cells. However, relatively few attentions have been paid to the effect of ADMA on monocytes, one of the important cells throughout all stages of atherosclerosis. In the present study, we found that reinioside C, the main component extracted from Polygala fallax Hemsl., dose-dependently inhibited tumor necrosis factor-α (TNF-α) production induced by ADMA in monocytes, Furthermore, reinioside C attenuated ADMA-induced generation of reactive oxygen species and activation of nuclear factor-κB (NF–κB) activity in monocytes in a dose-dependent manner, this effect was inhibited by l-arginine (NOS substrate) and PDTC (inhibitor of NF–κB). These data suggest that reinioside C could attenuate the increase of TNF-α induced by exogenous ADMA through inhibition ROS/NF–κB pathway in monocytes.

Nitric oxide mediates cardiac protection of tissue kallikrein by reducing inflammation and ventricular remodeling after myocardial ischemia/reperfusion

We assessed the role of nitric oxide (NO) and the kinin B2 receptor in mediating tissue kallikrein's actions in intramyocardial inflammation and cardiac remodeling after ischemia/reperfusion (I/R) injury. Adenovirus carrying the human tissue kallikrein gene was delivered locally into rat hearts 4 days prior to 30-minute ischemia followed by 24-hour or 7-day reperfusion with or without administration of icatibant, a kinin B2 receptor antagonist, or N(ω)–nitro–L–arginine methyl ester (L–NAME), a nitric oxide synthase inhibitor. Kallikrein gene delivery improved cardiac contractility and diastolic function, reduced infarct size at 1 day after I/R without affecting mean arterial pressure. Kallikrein treatment reduced macrophage/monocyte and neutrophil accumulation in the infarcted myocardium in association with reduced intercellular adhesion molecule-1 levels. Kallikrein increased cardiac endothelial nitric oxide synthase phosphorylation and NO levels and decreased superoxide formation, TGF–β1 levels and Smad2 phosphorylation. Furthermore, kallikrein reduced I/R-induced JNK, p38MAPK, IκB-α phosphorylation and nuclear NF–κB activation. In addition, kallikrein improved cardiac performance, reduced infarct size and prevented ventricular wall thinning at 7 days after I/R. The effects of kallikrein on cardiac function, inflammation and signaling mediators were all blocked by icatibant and L–NAME. These results indicate that tissue kallikrein through kinin B2 receptor and NO formation improves cardiac function, prevents inflammation and limits left ventricular remodeling after myocardial I/R by suppression of oxidative stress, TGF–β1/Smad2 and JNK/p38MAPK signaling pathways and NF–κB activation.

Redox State of Dipyridamole is a Critical Determinant for Its Beneficial Antioxidant and Antiinflammatory Effects

Dipyridamole, a well-known inhibitor of cGMP-dependent phosphodiesterase and the adenosine transporter, reportedly possesses antioxidant properties and attenuates reactive oxygen species (ROS) formation in platelet and endothelial cells. The relevance of the redox status of this compound or the mechanism for its redox-dependent effects is unknown. Oxidation of dipyridamole by horseradish peroxidase and hydrogen peroxide diminished its fluorescence and attenuated dipyridamole-mediated DPPH and ferric ferrozine reduction. Oxidation also led to elimination of dipyridamole's redox-sensitive properties, including inhibiting Cu (II)-induced LDL oxidation and ROS generation. Attenuation of activation- induced platelet release of soluble CD40 ligand (sCD40L) was diminished after dipyridamole oxidation. Dipyridamole but not oxidized dipyridamole effectively inhibited platelet adhesion to collagen-coated slides under flow conditions. By Western blot analysis, dipyridamole enhanced stimulation-induced platelet VASP phosphorylation, whereas oxidized dipyridamole caused attenuation. Using luciferase assays and nuclear translocation studies with confocal microscopy and Western blot analysis, native dipyridamole diminished TNF alpha or thrombin-induced NF kappa B activation and I kappa B alpha phosphorylation. Oxidized dipyridamole had no effect on TNFalpha-mediated NF kappa B activation. These results indicate: (1) the redox state of dipyridamole regulates its antioxidant properties; (2) dipyridamole's platelet inhibitory effects are manifested by enhanced VASP phosphorylation and platelet adhesion on collagen; and (3) dipyridamole's antioxidant effects in vascular cells are at least partially mediated via suppression of inflammatory NF kappa B signaling.

Binding of a venom Lys-49 phospholipase A 2 to LPS and suppression of its effects on mouse macrophages

The Lys49-phospholipases A 2 (K49-PLAs) are abundant in many pit vipers’ venom. They are highly basic myotoxins and capable of binding membranes but lack hydrolytic activity. Considerable attention has been directed to its antibacterial activity but the exact mechanisms remain unclear. We now evaluate the roles of a K49-PLA from Trimeresurus stejnegeri venom in antagonizing the effects of lipopolysaccharide (LPS) on mouse macrophages (RAW264.7 cells). The K49-PLA markedly reduced LPS-stimulated production of NO, MCP-1, RANTES, and iNOS. RT-PCR analysis also confirmed its suppression of LPS-induced transcription of these cellular proteins. Moreover, LPS-induced activation of NF κB was dramatically abolished, while phosphorylation and degradation of I κB were also inhibited. Other types of venom phospholipases tested did not show the same effects as K49-PLA. Finally, strong binding between K49-PLA and LPS with a dissociation constant at the order of 10 nM was shown by microcalorimetry titration. These findings provide unprecedented evidence that a low dose of K49-PLA possesses potent anti-inflammatory and antibacterial properties, which raises the prospect of a new therapeutic approach against sepsis.

Hypertension accelerated experimental abdominal aortic aneurysm through upregulation of nuclear factor κB and Ets

Conclusion: Hypertension accelerates progression of experimental abdominal aortic aneurysms (AAAs) by upregulation of Ets and nuclear factor κB (NF κB). Summary: The pathogenesis of AAA is complex but appears to involve upregulation of certain matrix metalloproteinases (MMPs). Experimental AAAs are characterized by upregulation of transcription factors, NF κB, which regulate transcription, and Ets, which regulates expression of MMPs. The role of hypertension in pathogenesis of AAA is controversial, with some studies suggesting it contributes to aneurysm formation and other studies suggesting it is not an independent risk factor for aneurysm formation. In this study, the authors used an experimental model of AAA to investigate the effects of hypertension on transcription factors NF κB and Ets. They also investigated the effects of hypertension on progression of AAA. Experimental AAAs were produced by elastase perfusion in both normotensive and hypertensive rats. Compared with normotensive rats, the size of the AAAs increased more rapidly in the hypertensive rats. Western blot analysis indicated the expression of MMP-2, MMP-3, MMP-9, and MMP-12, as well as that of intercellular adhesion molecule, was increased in hypertensive rats. This was accompanied by upregulation of NFκB and Ets. There was also increased activity of MMPs in the aortas of hypertensive rats vs those of normotensive rats. Chimeric decoy oligodeoxynucleotide (ODN) inhibits expression of NFκB and Ets. Transfection of the ODN resulted in significant inhibition of aortic dilatation in both hypertensive and normotensive rats. Transfection with chimeric decoy ODN resulted in significant inhibition of degeneration of elastic fibers in aortas of both normotensive and hypertensive rats. Expression of MMP-2, MMP-3, MMP-9, and MMP-12 and intercellular adhesion molecule was decreased by chimeric decoy ODN. This was accompanied by inhibition of macrophage migration. Comment: The study indicates hypertension accelerates progression of AAA in an experimental rat model. Perhaps inhibition of NF κB and Ets may someday be used as a method of decreasing aneurysm expansion in both normotensive and hypertensive patients.

Anti-inflammatory activity of flavonoids from Eupatorium arnottianum

Three anti-inflammatory compounds: nepetin, jaceosidin and hispidulin have been isolated and identified from Eupatorium arnottianum Griseb. dichloromethane extract. Nepetin reduced the TPA mouse ear edema by 46.9% and jaceosidin by 23.2% (1 mg/ear). Both compounds inhibited the NF κB induction by 91 and 77%, respectively. Furthermore phytochemical analysis of the ethanol extract has led to the identification of eriodictyol, hyperoside, rutin, caffeic and chlorogenic acids. All these compounds are reported for the first time in this species. The finding of topical antiinflammatory activity exerted by Eupatorium arnottianum extract and the identification of active principles could support the use of this plant for the treatment of inflammatory affections.

Cloning, expression and characterization of the bovine p65 subunit of NF κB

The full length coding sequence of the cattle transcription factor p65 was isolated and cloned. The cloned bovine p65 was expressed in mammalian cells, and it induced the NF-kappaB-specific luciferase reporter gene expression. Using gel retardation techniques, we demonstrated that the cloned bovine p65 bound to the consensus kappaB sequence. The comparison of the bovine p65 with its human and mouse orthologues at amino acid level showed high homology in both the DNA-binding domain, known as Rel homology domain (RHD) and the transactivation domain (TAD). The phylogenetic analysis at DNA level provided a new insight in the evolution of the NF-kappaB family, and it could resolve the topology of the mammalian p65 molecules. Although, the RHD was conserved in vertebrates, the TAD sequences deviated from each other, and showed faster molecular evolution than RHD sequences, which may indirectly result in the modification of NF-kappaB immune functions.

P3-406: Presenilin-1 interacts with tumor necrosis factor receptor-associated factor-6 (TRAF6): Implications in Alzheimer’s disease and cytokine responsiveness

The observation that presenilins are absolutely required for the pathogenesis of Alzheimer's disease (AD) and studies demonstrating that presenilins interact with several signalling molecules have led to the suggestion that involvement of presenilins in pathways other than presenilin–mediated cleavage of amyloid precursor protein may contribute to the onset of AD. We have identified a novel interaction between presenilins and members of the tumor necrosis factor receptor associated factor (TRAF) family of proteins. The TRAF adapter proteins (TRAF1–7) interact with members of the TNF, and Toll/IL–1 receptor super–families and are involved in several biological functions, including adaptive and innate immunity, control of programmed cell death within the developing CNS, stress response and bone metabolism. Using a bioinformatics and sequence analysis approach we have identified conserved TRAF–binding consensus sequences within presenilins. PS1 contains a TRAF6 binding domain, while both PS1 and PS2 contain a TRAF2–binding consensus. In transient transfection and co–immunoprecipitation experiments we show that PS1 co–immunoprecipitated with Flag–tagged TRAF2 and TRAF6 from HEK293 cell lysates. The minimal TRAF6 domain sufficient for PS1 binding was mapped to the N–terminal RING domain. Binding of PS1 to TRAF6 was mapped to the newly identified consensus sequence as mutating this site attenuates the interaction between PS1 and TRAF6. Co–expression of PS1 dramatically increased polyubiquitination of TRAF6 and enhanced TRAF6 induced NF–κB activation. Consistent with this and a role in neuroprotection, we show that in immortalized primary fibroblasts from PS1–/– mice, PS1 deficiency impairs responsiveness to cytokine–mediated activation of the NF–κB transcription factor. The identification of a novel interaction between presenilin–1 and TRAF6 may implicate the involvement of presenilin in downstream signaling events.