Reactivation of Epstein-Barr Virus by HIF-1α Requires p53.

We previously reported that the cellular transcription factor hypoxia-inducible factor 1α (HIF-1α) binds a hypoxia response element (HRE) located within the promoter of Epstein-Barr virus's (EBV's) latent-lytic switch BZLF1 gene, Zp, inducing viral reactivation. In this study, EBV-infected cell lines derived from gastric cancers and Burkitt lymphomas were incubated with HIF-1α-stabilizing drugs: the iron chelator deferoxamine (Desferal [DFO]), a neddylation inhibitor (pevonedistat [MLN-4924]), and a prolyl hydroxylase inhibitor (roxadustat [FG-4592]). DFO and MLN-4924, but not FG-4592, induced accumulation of both lytic EBV proteins and phosphorylated p53 in cell lines that contain a wild-type p53 gene. FG-4592 also failed to activate transcription from Zp in a reporter assay despite inducing accumulation of HIF-1α and transcription from another HRE-containing promoter. Unexpectedly, DFO failed to induce EBV reactivation in cell lines that express mutant or no p53 or when p53 expression was knocked down with short hairpin RNAs (shRNAs). Likewise, HIF-1α failed to activate transcription from Zp when p53 was knocked out by CRISPR-Cas9. Importantly, DFO induced binding of p53 as well as HIF-1α to Zp in chromatin immunoprecipitation (ChIP) assays, but only when the HRE was present. Nutlin-3, a drug known to induce accumulation of phosphorylated p53, synergized with DFO and MLN-4924 in inducing EBV reactivation. Conversely, KU-55933, a drug that inhibits ataxia telangiectasia mutated, thereby preventing p53 phosphorylation, inhibited DFO-induced EBV reactivation. Lastly, activation of Zp transcription by DFO and MLN-4924 mapped to its HRE. Thus, we conclude that induction of BZLF1 gene expression by HIF-1α requires phosphorylated, wild-type p53 as a coactivator, with HIF-1α binding recruiting p53 to Zp.IMPORTANCE EBV, a human herpesvirus, is latently present in most nasopharyngeal carcinomas, Burkitt lymphomas, and some gastric cancers. To develop a lytic-induction therapy for treating patients with EBV-associated cancers, we need a way to efficiently reactivate EBV into lytic replication. EBV's BZLF1 gene product, Zta, usually controls this reactivation switch. We previously showed that HIF-1α binds the BZLF1 gene promoter, inducing Zta synthesis, and HIF-1α-stabilizing drugs can induce EBV reactivation. In this study, we determined which EBV-positive cell lines are reactivated by classes of HIF-1α-stabilizing drugs. We found, unexpectedly, that HIF-1α-stabilizing drugs only induce reactivation when they also induce accumulation of phosphorylated, wild-type p53. Fortunately, p53 phosphorylation can also be provided by drugs such as nutlin-3, leading to synergistic reactivation of EBV. These findings indicate that some HIF-1α-stabilizing drugs may be helpful as part of a lytic-induction therapy for treating patients with EBV-positive malignancies that contain wild-type p53.

Hypoxia-inducible factor-1α plays roles in Epstein-Barr virus's natural life cycle and tumorigenesis by inducing lytic infection through direct binding to the immediate-early BZLF1 gene promoter.

When confronted with poor oxygenation, cells adapt by activating survival signaling pathways, including the oxygen-sensitive transcriptional regulators called hypoxia-inducible factor alphas (HIF-αs). We report here that HIF-1α also regulates the life cycle of Epstein-Barr virus (EBV). Incubation of EBV-positive gastric carcinoma AGS-Akata and SNU-719 and Burkitt lymphoma Sal and KemIII cell lines with a prolyl hydroxylase inhibitor, L-mimosine or deferoxamine, or the NEDDylation inhibitor MLN4924 promoted rapid and sustained accumulation of both HIF-1α and lytic EBV antigens. ShRNA knockdown of HIF-1α significantly reduced deferoxamine-mediated lytic reactivation. HIF-1α directly bound the promoter of the EBV primary latent-lytic switch BZLF1 gene, Zp, activating transcription via a consensus hypoxia-response element (HRE) located at nt -83 through -76 relative to the transcription initiation site. HIF-1α did not activate transcription from the other EBV immediate-early gene, BRLF1. Importantly, expression of HIF-1α induced EBV lytic-gene expression in cells harboring wild-type EBV, but not in cells infected with variants containing base-pair substitution mutations within this HRE. Human oral keratinocyte (NOK) and gingival epithelial (hGET) cells induced to differentiate by incubation with either methyl cellulose or growth in organotypic culture accumulated both HIF-1α and Blimp-1α, another cellular factor implicated in lytic reactivation. HIF-1α activity also accumulated along with Blimp-1α during B-cell differentiation into plasma cells. Furthermore, most BZLF1-expressing cells observed in lymphomas induced by EBV in NSG mice with a humanized immune system were located distal to blood vessels in hypoxic regions of the tumors. Thus, we conclude that HIF-1α plays central roles in both EBV's natural life cycle and EBV-associated tumorigenesis. We propose that drugs that induce HIF-1α protein accumulation are good candidates for development of a lytic-induction therapy for treating some EBV-associated malignancies.

Drugs That Mimic Hypoxia Selectively Target EBV-Positive Gastric Cancer Cells.

Latent infection of Epstein-Barr virus (EBV) is associated with lymphoid and epithelial cell cancers, including 10% of gastric carcinomas. We previously reported that hypoxia inducible factor-1α (HIF-1α) induces EBV's latent-to-lytic switch and identified several HIF-1α-stabilizing drugs that induce this viral reactivation. Here, we tested three classes of these drugs for preferential killing of the EBV-positive gastric cancer AGS-Akata cell line compared to its matched EBV-negative AGS control. We observed preferential killing with iron chelators [Deferoxamine (DFO); Deferasirox (DFX)] and a prolyl hydroxylase inhibitor (BAY 85-3934 (Molidustat)), but not with a neddylation inhibitor [MLN4924 (Pevonedistat)]. DFO and DFX also induced preferential killing of the EBV-positive gastric cancer AGS-BDneo and SNU-719 cell lines. Preferential killing was enhanced when low-dose DFX (10 µM) was combined with the antiviral prodrug ganciclovir. DFO and DFX induced lytic EBV reactivation in approximately 10% of SNU-719 and 20-30% of AGS-Akata and AGS-BDneo cells. However, neither DFO nor DFX significantly induced synthesis of lytic EBV proteins in xenografts grown in NSG mice from AGS-Akata cells above the level observed in control-treated mice. Therefore, these FDA-approved iron chelators are less effective than gemcitabine at promoting EBV reactivation in vivo despite their high specificity and efficiency in vitro.

Laser vibrometry from a moving ground vehicle.

We investigated the fundamental limits to the performance of a laser vibrometer that is mounted on a moving ground vehicle. The noise floor of a moving laser vibrometer consists of speckle noise, shot noise, and platform vibrations. We showed that speckle noise can be reduced by increasing the laser spot size and that the noise floor is dominated by shot noise at high frequencies (typically greater than a few kilohertz for our system). We built a five-channel, vehicle-mounted, 1.55 µm wavelength laser vibrometer to measure its noise floor at 10 m horizontal range while driving on dirt roads. The measured noise floor agreed with our theoretical estimates. We showed that, by subtracting the response of an accelerometer and an optical reference channel, we could reduce the excess noise (in units of micrometers per second per Hz(1/2)) from vehicle vibrations by a factor of up to 33, to obtain nearly speckle-and-shot-noise-limited performance from 0.3 to 47 kHz.

Structure of the calcium-rich signature domain of human thrombospondin-2.

Thrombospondins (THBSs) are secreted glycoproteins that have key roles in interactions between cells and the extracellular matrix. Here, we describe the 2.6-A-resolution crystal structure of the glycosylated signature domain of human THBS2, which includes three epidermal growth factor-like modules, 13 aspartate-rich repeats and a lectin-like module. These elements interact extensively to form three structural regions termed the stalk, wire and globe. The THBS2 signature domain is stabilized by these interactions and by a network of 30 bound Ca(2+) ions and 18 disulfide bonds. The structure suggests how genetic alterations of THBSs result in disease.

The anatomic site of the transepithelial permeability barriers of toad bladder.

An examination of the mucosal epithelium of the urinary bladder of the toad reveals that the two major cell types which abut on the urinary surface, the granular and mitochondria-rich cells, also contact the basement membrane. Thus, the epithelium functions as a single cell layer. Although basal cells are interpolated between the granular cells and the basement membrane over a large portion of the epithelium, they do not constitute an additional continuous cell layer. This finding is consistent with extensive physiological data which had assumed that the major permeability barriers of this epithelium were the apical and basal-lateral plasma membranes of a single layer of cells.

Fibroblast growth factor-2 binding to the thrombospondin-1 type III repeats, a novel antiangiogenic domain.

Thrombospondin-1, an antiangiogenic matricellular protein, binds with high affinity to the angiogenic fibroblast growth factor-2, affecting its bioavailability and activity. The present work aimed at further locating the fibroblast growth factor-2 binding site of thrombospondin-1 and investigating its activity, using recombinant thrombospondin-1 proteins. Only recombinant constructs containing the thrombospondin-1 type III repeats bound fibroblast growth factor-2, whereas other domains, including the known anti-angiogenic type I repeats, were inactive. Binding was specific and inhibited by the anti thrombospondin-1 monoclonal antibody B5.2. Surface plasmon resonance analysis on BIAcore revealed a binding affinity (K(d)) of 310nM for the type III repeats and 11nM for intact thrombospondin-1. Since the type III repeats bind calcium, the effect of calcium on thrombospondin-1 binding to fibroblast growth factor-2 was investigated. Binding was modulated by calcium, as thrombospondin-1 or the type III repeats bound to fibroblast growth factor-2 only in calcium concentrations <0.3mM. The type III repeats inhibited binding of fibroblast growth factor-2 to endothelial cells, fibroblast growth factor-2-induced endothelial cell proliferation in vitro and angiogenesis in the chorioallantoic membrane assay in vivo, thus indicating the antiangiogenic activity of the domain. In conclusion, this study demonstrates that the fibroblast growth factor-2 binding site of thrombospondin-1 is located in the type III repeats. The finding that this domain is active in inhibiting angiogenesis indicates that the type III repeats represent a novel antiangiogenic domain of thrombospondin-1.

The role of cell swelling in ischemic renal damage and the protective effect of hypertonic solute.

The failure of blood flow to return to the kidney following a transient period of ischemia has long been recognized. The cause of this "no-reflow" has been investigated in the rat after a transient period of total obstruction of the renal arteries. The vascular pattern of the kidneys as visualized with silicone rubber injection shows a diffuse patchy ischemia throughout the kidney, which persists after release of the obstructed renal artery. Electron microscopic studies of ischemic kidneys showed that all cellular elements were swollen and limiting the available vascular space. Functional studies revealed an increase in plasma urea nitrogen and creatinine after 1 hr or longer ischemic periods. The ischemia, cell swelling, "no-reflow," and subsequent renal dysfunction occurring after obstruction to the renal arteries were corrected by the administration of hypertonic mannitol, but were unaffected by an equivalent expansion of the extracellular fluid volume either with isotonic saline or isotonic mannitol, showing that the osmotic effect was primary. The hypothesis is presented that ischemic swelling of cells may occlude small blood vessels so that recirculation does not resume even after the initial cause of the ischemia is no longer present; solutes which do not penetrate cell membranes are able to shrink swollen cells, increase the available vascular space and thus permit reflow of blood to the ischemic organ.

Improvement in myocardial function and coronary blood flow in ischemic myocardium after mannitol.

The purpose of this study was to evaluate the effect of hyperosmolality on the performance of, and the collateral blood flow to, ischemic myocardium. The myocardial response to mannitol, a hyperosmolar agent which remains extracellular, was evaluated in anesthetized dogs. Mannitol was infused into the aortic roots of 31 isovolumic hearts and of 15 dogs on right heart bypass, before and during ischemia. Myocardial ischemia was produced by temporary ligation of either the proximal or mid-left anterior descending coronary artery. Mannitol significantly improved the depressed ventricular function curves which occurred with left anterior descending coronary artery occlusion. Mannitol also significantly lessened the S-T segment elevation (epicardial electrocardiogram) occurring during myocardial ischemia in the isovolumic hearts and this reduction was associated with significant increases in total coronary blood flow (P < 0.005) and with increased collateral coronary blood flow to the ischemia area (P < 0.005).THUS, INCREASES IN SERUM OSMOLALITY PRODUCED BY MANNITOL RESULT IN THE FOLLOWING BENEFICIAL CHANGES DURING MYOCARDIAL ISCHEMIA: (a) improved myocardial function, (b) reduced S-T segment elevation, (c) increased total coronary blood flow, and (d) increased collateral coronary blood flow.

Prevention of sudden cardiac death by dietary pure omega-3 polyunsaturated fatty acids in dogs.

BACKGROUND: Rat diets high in fish oil have been shown to be protective against ischemia-induced fatal ventricular arrhythmias. Increasing evidence suggests that this may also apply to humans. To confirm the evidence in animals, we tested a concentrate of the free fish-oil fatty acids and found them to be antiarrhythmic. In this study, we tested the pure free fatty acids of the 2 major dietary omega-3 polyunsaturated fatty acids in fish oil: cis-5,8,11,14, 17-eicosapentaenoic acid (C20:5omega-3) and cis-4,7,10,13,16, 19-docosahexaenoic acid (C22:6omega-3), and the parent omega-3 fatty acid in some vegetable oils, cis-9,12,15-alpha-linolenic acid (C18:3omega-3), administered intravenously on albumin or a phospholipid emulsion. METHODS AND RESULTS: The tests were performed in a dog model of cardiac sudden death. Dogs were prepared with a large anterior wall myocardial infarction produced surgically and an inflatable cuff placed around the left circumflex coronary artery. With the dogs running on a treadmill 1 month after the surgery, occlusion of the left circumflex artery regularly produced ventricular fibrillation in the control tests done 1 week before and after the test, with the omega-3 fatty acids administered intravenously as their pure free fatty acid. With infusion of the eicosapentaenoic acid, 5 of 7 dogs were protected from fatal ventricular arrhythmias (P<0.02). With docosahexaenoic acid, 6 of 8 dogs were protected, and with alpha-linolenic acid, 6 of 8 dogs were also protected (P<0.004 for each). The before and after control studies performed on the same animal all resulted in fatal ventricular arrhythmias, from which they were defibrillated. CONCLUSIONS: These results indicate that purified omega-3 fatty acids can prevent ischemia-induced ventricular fibrillation in this dog model of sudden cardiac death.

Infusional cyclophosphamide, doxorubicin, and etoposide in relapsed and resistant non-Hodgkin's lymphoma: evidence for a schedule-dependent effect favoring infusional administration of chemotherapy.

PURPOSE: This study attempted to determine the efficacy of cyclophosphamide (C), doxorubicin (D), and etoposide (E) administered as a continuous intravenous (IV) infusion (infusional CDE) over 4 days in patients with relapsed or resistant non-Hodgkin's lymphoma (rNHL) and in patients with previously untreated (uNHL) who had poor prognostic features. PATIENTS AND METHODS: Fifty-eight patients with rNHL and 10 patients with uNHL received infusional CDE every 28 or more days; all but one had intermediate- to high-grade histology. The cumulative doses of C, D, and E administered per treatment cycle were 750 mg/m2, 50 mg/m2, and 240 mg/m2, respectively. In the rNHL group, all patients had previously received C, most (81%) had received D, and a minority (16%) had received E. RESULTS: Objective response occurred in 30 patients with rNHL (52%; 95% confidence interval, 39% to 65%); 10 patients had a complete response (CR) (17%; 95% confidence interval, 7% to 27%). Eleven patients (19%) remain progression-free (median follow-up, 22 months; range, 10+ to 38+), and six patients (10%) are disease-free (median follow-up, 25 months; range, 10+ to 38+). Among 10 patients with uNHL, eight (80%) had a CR, and none have relapsed (median follow-up, 11 months; range, 9+ to 24+). Toxicity was primarily hematologic. Two treatment-related deaths (3%) occurred, both attributable to infection in the relapsed or resistant group. CONCLUSION: Infusional CDE produced a CR in substantial proportion of patients who had previously been exposed to at least two of the agents administered as an IV bolus, suggesting a schedule-dependent effect in favor of the infusional administration of certain cytotoxic agents in patients with lymphoid neoplasms. In addition, infusional CDE was effective and tolerable in patients with poor-prognosis NHL when used as initial therapy, and merits further study in that setting.

The relation between salt and ionic transport coefficients.

The reflection coefficient was originally introduced by Staverman to describe the movement of nonelectrolytes through membranes. When this coefficient is extended to salts, one has a choice of defining this term for the whole salt moving as a single electrically neutral component or for the individual ions of the salt. The latter definition is meaningful only in the absence of an electric field across the permeability barrier. This condition may be achieved with the voltage clamp or short-circuit technique and is especially useful in dealing with biological systems in which one rarely has only a single salt or even equal concentrations of the major anion and cation. The relations between the transport coefficients for the salt and its individual ions are derived. The special conditions which may result in negative osmosis through a charged membrane in the presence of a salt are discussed.

Feeding growth restricted preterm infants with abnormal antenatal Doppler results.

Absence or reversal of end diastolic flow (AREDF) in the umbilical artery is associated with poor outcome, and elective premature delivery is common. Feeding these infants is a challenge. They often have poor tolerance of enteral feeding, and necrotising enterocolitis may develop. This review explores current practice to see if there is evidence on which to base guidelines. The incidence of necrotising enterocolitis is increased in infants with fetal AREDF, especially when complicated by fetal growth restriction. Abnormalities of splanchnic blood flow persist postnatally, with some recovery during the first week of life, providing justification for a delayed and careful introduction of enteral feeding. Such a policy exposes babies to the risks of parenteral nutrition, with no trials to date showing any benefit of delayed enteral nutrition. Trials are required to determine the optimum timing for introduction of enteral feeds in growth restricted infants with fetal AREDF.

Ten years of disease-free survival between two diagnoses of small-cell lung cancer: a case report and a literature review.

Small-cell lung carcinoma (SCLC) represents one-fifth of all cases of bronchopulmonary cancer and has a 5-yr survival of 2-4%. Long-term survivors of SCLC are at risk for developing second primary aerodigestive tumors. We report a case of a long-term survivor who had a 10-yr disease-free survival between two diagnoses of SCLC. A literature review identified four case reports and seven review series with a total of 26 cases of 5-yr disease-free survivors of SCLC who developed a second SCLC. A total of 4574 patients were reported in the review series. Five-year disease-free survival was documented in 139 of 4574 patients. Twenty-two (15.8%) of those developed a second SCLC over the next 7 yr of follow up, with an averaged annual incidence equal to fivefold that of the general population. Earlier reports of a slow radiographic doubling time for some cases of SCLC suggests that survivors of SCLC may still develop a recurrent SCLC following 12 yr of disease-free follow up. It remains difficult to ascertain whether a second SCLC is a recurrence or a second primary tumor in the absence of a preneoplastic lesion for SCLC. New genetic markers may hold the answer. They may also help screen high-risk patients including survivors of SCLC.

Prevention of fatal cardiac arrhythmias by polyunsaturated fatty acids.

In animal feeding studies, and probably in humans, n-3 polyunsaturated fatty acids (PUFAs) prevent fatal ischemia-induced cardiac arrhythmias. We showed that n-3 PUFAs also prevented such arrhythmias in surgically prepared, conscious, exercising dogs. The mechanism of the antiarrhythmic action of n-3 PUFAs has been studied in spontaneously contracting cultured cardiac myocytes of neonatal rats. Adding arrhythmogenic toxins (eg, ouabain, high Ca(2+), lysophosphatidylcholine, beta-adrenergic agonist, acylcarnitine, and the Ca(2+) ionophore) to the myocyte perfusate caused tachycardia, contracture, and fibrillation of the cultured myocytes. Adding eicosapentaenoic acid (EPA: 5-15 micromol/L) to the superfusate before adding the toxins prevented the expected tachyarrhythmias. If the arrhythmias were first induced, adding the EPA to the superfusate terminated the arrhythmias. This antiarrhythmic action occurred with dietary n-3 and n-6 PUFAs; saturated fatty acids and the monounsaturated oleic acid induced no such action. Arachidonic acid (AA; 20:4n-6) is anomalous because in one-third of the tests it provoked severe arrhythmias, which were found to result from cyclooxygenase metabolites of AA. When cyclooxygenase inhibitors were added with the AA, the antiarrhythmic effect was like those of EPA and DHA. The action of the n-3 and n-6 PUFAs is to stabilize electrically every myocyte in the heart by increasing the electrical stimulus required to elicit an action potential by approximately 50% and prolonging the relative refractory time by approximately 150%. These electrophysiologic effects result from an action of the free PUFAs to modulate sodium and calcium currents in the myocytes. The PUFAs also modulate sodium and calcium channels and have anticonvulsant activity in brain cells.

Psychological, behavioral and biochemical risk factors for coronary artery disease among American and Italian male corporate managers.

Differences in psychological, behavioral and biochemical risk factors for coronary artery disease (CAD) among male corporate managers of 2 countries (United States and Italy), with very different age-specific rates of mortality for CAD were evaluated. In all, 129 American (mean age 43 +/- 7 years) and 80 Italian (mean age 45 +/- 7 years) managers volunteered to participate in this study. Each subject was administered several questionnaires assessing various psychological and behavioral risk factors for CAD, and all 129 Americans and 55 of 80 Italians had their blood drawn between 8:00 and 9:30 AM after overnight fasting for the measurement of plasma levels of dehydroepiandrosterone-sulfate (DHEA-S), total cholesterol, triglycerides, and apolipoproteins A-I and B. Italian managers reported significantly more cynicism and hostility, and less enjoyment in leisure activities than did American ones. Furthermore, 40 Italian (51%) and only 18 American (14%) managers were smokers (this difference being statistically significant). Although no significant differences were found in factors positively related with CAD (cholesterol, triglycerides and apolipoprotein B), there were clear differences in parameters inversely correlated with the incidence of CAD. Italian managers had significantly lower levels of plasma DHEA-S and apolipoprotein A-I than did American ones. In conclusion, this study found that Italian managers had a significantly more unhealthy psychological and behavioral profile than did American ones, and had lower levels of those biochemical parameters (apolipoprotein A-I and DHEA-S) thought to have a protective role against development of CAD.

Brain uptake and utilization of fatty acids: applications to peroxisomal biogenesis diseases.

The brain is rich in diverse fatty acids saturated, monounsaturated and polyunsaturated fatty acids with chain lengths ranging from less than 16 to more than 24 carbons that make up the complex lipids present in this organ. While some fatty acids are derived from endogenous synthesis, others must come from exogenous sources. The mechanism(s) by which fatty acids enter cells has been the subject of much debate. While some investigators argue for a protein-mediated process, others suggest that simple diffusion is sufficient. In the brain, uptake is further complicated by the presence of the blood-brain barrier. Brain fatty acid homeostasis is disturbed in many human disorders, as typified by the peroxisomal biogenesis diseases. A workshop designed to bring together researchers from varied backgrounds to discuss these issues in an open forum was held in March, 2000. In addition to assessing the current state of knowledge, areas requiring additional investigation were identified and recommendations for future research were made. A brief overview of the invited talks is presented here.

Omega-3 fatty acids and prevention of ventricular fibrillation.

Interest in the potential cardiovascular benefits of omega-3 long chain polyunsaturated fatty acids has been largely focused on possible antiatherothrombotic effects. In addition, however, definitive antiarrhythmic effects of these dietary omega-3 fatty acids have been reported by Charnock & McLennan. Our studies commenced with the observation that two of these fatty acids, eicosapentaenoic (C20:5n-3, EPA) and docosahexaenoic acid (C22:6n-3, DHA) prevented contracture and fibrillation of isolated neonatal cardiac myocytes when exposed to toxic levels of ouabain (0.1 mM). This protection was associated with prevention of excessively high intracellular calcium concentrations in the myocyte. Further, it was shown that these fatty acids modulate calcium currents through L-type calcium channels and that the effect occurs within a few minutes of adding EPA or DHA to the medium perfusing the cultured cardiac myocytes. Infusing an emulsion of the omega-3 fatty acids intravenously just prior to compression of a coronary artery in a conscious, prepared dog will prevent the expected subsequent ischemia-induced ventricular fibrillation.

Interactions of n-3 fatty acids with ion channels in excitable tissues.

In summary, we have shown that the conventional explanation for the site of action of a ligand which alters the conductance of a membrane ion channel is that the ligand interacts or binds with the ion channel protein, changing its conductance, is inadequate to explain the primary site of action of the antiarrhythmic n-3 PUFAs. We have shown that when a neutral asparagine is replaced by a positively charged lysine in the N406 amino acid site in the alpha-subunit of the human cardiac sodium channel, the n-3 fatty acids lose their inhibitory action on the sodium current. The inadequacy of this finding to explain the primary site of action of the n-3 PUFAs is demonstrated by the inhibitory effect on all other cardiac ion channels, so far tested. We show that ion channels, which share no amino acid homology with the PUFAs, have their conductance also reduced in the presence of the PUFAs, Thus a more general conceptual framework or paradigm is needed to account for the broad action of the PUFAs on diverse different ion channels lacking amino acid homology. We have been testing the membrane tension hypothesis of Andersen and associates. According to this hypothesis, the fatty acids are not acting directly on the ion channel protein but accumulating in the phospholipid membrane in immediate juxtaposition to the site in the membrane where the ion channel protein penetrates the membrane phospholipid bilayer. This alters membrane tensions exerted by the phospholipid membrane on the ion channel, which in turn causes conformational changes in the ion channel, altering the conductance of the ion channel. Our preliminary data seem to support this membrane tension hypothesis.