Cyclooxygenase (COX)-3, a novel COX splice variant, was suggested as the key to unlocking the mystery of the mechanism of action of acetaminophen. Although COX-3 might have COX activity in canines, and this activity might be inhibited by acetaminophen, its low expression level and the kinetics indicate unlikely clinical relevance. In rodents and humans, COX-3 encodes proteins with completely different amino acid sequences than COX

We investigated the effect of diazoxide on neuronal survival in primary cultures of rat cortical neurons against oxygenglucose deprivation (OGD). Diazoxide pre-treatment induced delayed pre-conditioning and almost entirely attenuated the OGD-induced neuronal death. Diazoxide inhibited succinate dehydrogenase and induced mitochondrial depolarization, free radical production and protein kinase C activation. The putative mitochondrial ATP-sensitive potassium channel blocker 5-hydroxydecanoate abolished the protective effect of diazoxide while the non-selective K ATP channel blocker glibenclamide did not. The non-selective K ATP channel openers nicorandil and cromakalim did not improve viability. Superoxide dismutase mimetic, M40401, or protein kinase C inhibitor, chelerythrine, prevented the neuroprotective effect of diazoxide. Diazoxide did not increase reduced glutathione and manganese-superoxide dismutase levels but we found significantly higher reduced glutathione levels in diazoxidepre-conditioned neurons after OGD. In pre-conditioned neurons free radical production was reduced upon glutamate stimulation. The succinate dehydrogenase inhibitor 3-nitropropionic acid also induced pre-conditioning and free radical production in neurons. Here, we provide the first evidence that diazoxide induces delayed pre-conditioning in neurons via acute generation of superoxide anion and activation of protein kinases and subsequent attenuation of oxidant stress following OGD. The succinate dehydrogenase-inhibiting effect of diazoxide is more likely to be involved in this neuroprotection than the opening of mitochondrial ATP-sensitive potassium channels.

No abstract

Flagella from diverse gram-negative bacteria induce tumor necrosis factor alpha (TNF-␣) and interleukin-1␤ (IL-1␤A large number of studies have demonstrated that multiple components of gram-negative and gram-positive bacteria possess the ability to stimulate the release of proinflammatory cytokines from monocytes and macrophages (4). These cytokine inducers have collectively been termed bacterial modulins. Lipopolysaccharide (LPS), the best-studied bacterial modulin from gram-negative organisms, has been shown to induce the synthesis of tumor necrosis factor alpha (TNF-␣) and other cytokines in vivo and in vitro. Although TNF-␣ plays an essential role in resistance to bacterial infections, it is also a major contributor to the deleterious effects leading to septic shock. More recently, other bacterial modulins have been identified that elicit the production of proinflammatory cytokines (4). These include lipoteichoic acid and peptidoglycan from gram-positive organisms, lipoarabinomannan from mycobacteria, lipoproteins from mycoplasmas, and porins from gramnegative organisms. We (1, 3) and others (14) have shown that flagella or small fragments of flagella from several species of gram-negative bacteria stimulate TNF-␣ and interleukin-1␤ synthesis in human peripheral blood mononuclear cells (PBMC). Using genetic and biochemical approaches, we demonstrated that the expression of the major flagellar filament subunit, flagellin, is required for cytokine induction by gramnegative organisms (1).Gram-negative bacteria such as Escherichia coli, Salmonella enterica serovar Enteritidis, and Pseudomonas aeruginosa produce flagellins with molecular masses of approximately 40 to 60 kDa (6). For example, the salmonella flagellins have a molecular mass of approximately 50 kDa. Alignment of amino acid sequences from different gram-negative species shows a high degree of sequence similarity in the amino-and carboxy-terminal regions, comprising approximately the N-terminal 150 and C-terminal 85 residues of the protein. In contrast, the central hypervariable regions of these proteins are quite divergent. Differences in length within the hypervariable domains account for most of the variation in molecular mass among different species.Although flagellin expression is essential for the TNF-␣-inducing activity of flagella (1), it was thought possible that the role of flagellin is simply to stabilize the actual inducer and not to induce cytokine synthesis. For example, the flagellin may be required to present FliD, the flagellum cap protein. To address this question, we prepared purified recombinant flagellins from S. enterica serovar Enteritidis and S. enterica serovar Typhimurium as well as P. aeruginosa and tested each protein for TNF-␣-inducing activity in cultures of PBMC and THP-1 cells, a human myelomonocytic cell line. Using deletion mutants of flagellin, we defined the region(s) of the flagellin protein required for TNF-␣-inducing activity. In addition, we evaluated the possibility that flagellin induces cytokine production in ...

Insulin resistance (IR) precedes the onset of Type 2 diabetes, but its impact on preconditioning against myocardial ischemia-reperfusion injury is unexplored. We examined the effects of diazoxide and ischemic preconditioning (IPC; 5-min ischemia and 5-min reperfusion) on ischemia (30 min)-reperfusion (240 min) injury in young IR Zucker obese (ZO) and lean (ZL) rats. ZO hearts developed larger infarcts than ZL hearts (infarct size: 57.3 +/- 3% in ZO vs. 39.2 +/- 3.2% in ZL; P < 0.05) and also failed to respond to cardioprotection by IPC or diazoxide (47.2 +/- 4.3% and 52.5 +/- 5.8%, respectively; P = not significant). In contrast, IPC and diazoxide treatment reduced the infarct size in ZL hearts (12.7 +/- 2% and 16.3 +/- 6.7%, respectively; P < 0.05). The mitochondrial ATP-activated potassium channel (K(ATP)) antagonist 5-hydroxydecanoic acid inhibited IPC and diazoxide-induced preconditioning in ZL hearts, whereas it had no effect on ZO hearts. Diazoxide elicited reduced depolarization of isolated mitochondria from ZO hearts compared with ZL (73 +/- 9% in ZL vs. 39 +/- 9% in ZO; P < 0.05). Diazoxide also failed to enhance superoxide generation in isolated mitochondria from ZO compared with ZL hearts. Electron micrographs of ZO hearts revealed a decreased number of mitochondria accompanied by swelling, disorganized cristae, and vacuolation. Immunoblots of mitochondrial protein showed a modest increase in manganese superoxide dismutase in ZO hearts. Thus obesity accompanied by IR is associated with the inability to precondition against ischemic cardiac injury, which is mediated by enhanced mitochondrial oxidative stress and impaired activation of mitochondrial K(ATP).

Reduced susceptibility to infectious disease can increase the frequency of otherwise deleterious alleles. In populations of African ancestry, two apolipoprotein-L1 (APOL1) variants with a recessive kidney disease risk, named G1 and G2, occur at high frequency. APOL1 is a trypanolytic protein that confers innate resistance to most African trypanosomes, but not Trypanosoma brucei rhodesiense or T.b. gambiense, which cause human African trypanosomiasis. In this case-control study, we test the prevailing hypothesis that these APOL1 variants reduce trypanosomiasis susceptibility, resulting in their positive selection in sub-Saharan Africa. We demonstrate a five-fold dominant protective association for G2 against T.b. rhodesiense infection. Furthermore, we report unpredicted strong opposing associations with T.b. gambiense disease outcome. G2 associates with faster progression of T.b. gambiense trypanosomiasis, while G1 associates with asymptomatic carriage and undetectable parasitemia. These results implicate both forms of human African trypanosomiasis in the selection and persistence of otherwise detrimental APOL1 kidney disease variants.

Although APOL1 gene variants are associated with nephropathy in African Americans, little is known about APOL1 protein synthesis, uptake, and localization in kidney cells. To address these questions, we examined APOL1 protein and mRNA localization in human kidney and human kidney-derived cell lines. Indirect immunofluorescence microscopy performed on nondiseased nephrectomy cryosections from persons with normal kidney function revealed that APOL1 protein was markedly enriched in podocytes (colocalized with synaptopodin and Wilms' tumor suppressor) and present in lower abundance in renal tubule cells. Fluorescence in situ hybridization detected APOL1 mRNA in glomeruli (podocytes and endothelial cells) and tubules, consistent with endogenous synthesis in these cell types. When these analyses were extended to renal-derived cell lines, quantitative RT-PCR did not detect APOL1 mRNA in human mesangial cells; however, abundant levels of APOL1 mRNA were observed in proximal tubule cells and glomerular endothelial cells, with lower expression in podocytes. Western blot analysis revealed corresponding levels of APOL1 protein in these cell lines. To explain the apparent discrepancy between the marked abundance of APOL1 protein in kidney podocytes observed in cryosections versus the lesser abundance in podocyte cell lines, we explored APOL1 cellular uptake. APOL1 protein was taken up readily by human podocytes in vitro but was not taken up efficiently by mesangial cells, glomerular endothelial cells, or proximal tubule cells. We hypothesize that the higher levels of APOL1 protein in human cryosectioned podocytes may reflect both endogenous protein synthesis and APOL1 uptake from the circulation or glomerular filtrate.

Flagellin from a number of Gram-negative bacteria induces cytokine and nitric oxide production by inflammatory cell types. In view of the evidence that flagellin responsiveness is subject to modulation, we explored the possibilities that a prior exposure to flagellin might result in a state of reduced flagellin responsiveness or tolerance and that lipopolysaccharide ( Evidence from a large number of studies demonstrates the importance of cytokine production in the protective response against Gram-negative pathogens. We (1, 2) and others (3) reported that flagella from Gram-negative bacteria such as Salmonella enteritidis and Pseudomonas aeruginosa induce cytokine production (e.g. TNF-␣ 1 and IL-1) by human monocytes. Genetic evidence pointed to a role for flagellin, the major structural protein of the flagellum (1). Subsequently, we demonstrated that purified recombinant flagellin is an extraordinarily potent inducer of cytokine and nitric oxide production by monocytes (4, 5). Half-maximal stimulation of monocytes and THP1 cells was achieved with picomolar concentrations of flagellin. The proinflammatory action of flagellin has been confirmed by other investigators using Caco-2 cells and model epithelial systems (6 -12).Flagellin, like lipopolysaccharide (LPS) (13-16), signals via a toll-like receptor (TLR)/IL-1 receptor-associated kinase (IRAK)-dependent pathway (5,8,17). LPS signals via TLR4, whereas flagellin utilizes TLR5 (8, 17). The activation of IRAK by flagellin is a relatively rapid process; maximal activation of IRAK activity in human and murine monocytes occurs within 5-10 min after the addition of flagellin to the cells (5).In vitro studies with cultured monocytes and neutrophils as well as analysis of these cell types from patients with Gramnegative sepsis have established that prior exposure to LPS induces a transient state of cellular tolerance to subsequent stimulation by LPS (see e.g. . LPS tolerance is present within hours after an initial exposure to LPS (21-24). LPS tolerance may have evolved as a mechanism to limit the mediator storm that is induced by LPS and which is responsible for the pathologic events associated with LPS-induced septic shock. Because of the potential importance of LPS tolerance in the host response to Gram-negative infection, the underlying mechanism(s) that govern the induced state of LPS tolerance continues to be a subject of intense investigation. Although the mechanism for this effect has not been established definitively, evidence has been obtained and arguments made in favor of a role for a labile repressor (22), secretory leukocyte protease inhibitor (25), the down-regulation of TLR4 expression (26) and decreased IRAK expression (27). Investigators have also analyzed the potential for LPS, lipoarabinomannan (from mycobacteria) and lipopeptides (from mycoplasma) to induce a state of cross-tolerance to each other. The available evidence indicates that LPS can induce tolerance to several unrelated inducers including lipoarabinomannan (28) mycoplasma lipopeptides (29...