Enterotoxigenic strains of Staphylococcus aureus produce a variety of heat-stable staphylococcal enterotoxins (SEs) that are a prevalent cause of food poisoning in the United States and other countries. Many immunological and biochemical assays often work well in buffer systems but are hindered when tested in the complex chemical environment of foods. To overcome these biases and improve the limits of detection, we implemented an immunomagnetic PCR signal amplification assay (iPCR-SA) for recovery and detection of SEA and SEB in foods. Anti-SEA or anti-SEB primary antibodies were coated onto COOH-modified magnetic beads using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide reagent. Secondary antibodies were covalently linked to amino-modified reporter DNA oligonucleotides (563 bp) via the linker molecule succinimidyl-4[N-maleimidomethyl]-cyclohexane-1-carboxylate. An internal 159-bp portion of the reporter DNA retained by the captured toxin molecule was then amplified by real-time PCR. A semiautomated Bead Retriever proved extremely helpful in both the application of the conjugation chemistries and required washes and the recovery and washing of bead-conjugated toxin from tested food samples. The procedure was simple, and analyses were completed in 5 to 6 h. The assay was sufficiently robust that we were able to detect SEA and SEB in tryptic soy broth, milk, lemon cream pie, tuna salad, deli turkey, and ground turkey at levels as low as 7.5 fg/ml. SE was still detected at high sensitivity after heating in food samples for typical pasteurization or cooking regimens. Sensitivity was diminished only when samples were subjected to extreme heating.
We hypothesized that biological metabolites of quercetin, resveratrol, and grape seed extract previously identified in human plasma can prevent impairment of nitric oxide (NO) bioavailability due to glucotoxic conditions (e.g. Type 1 or 2 diabetes). Human aortic endothelial cells were treated for 24 h with 2μΜ Quercetin‐3‐O‐glucoronide, 5μΜ Piceatannol, or 1μΜ 3‐Hydroproponoic acid. Cells were next exposed to normal (5mM) or high (25mM) glucose for 48h, then treated with insulin (100nM, 10 min) to stimulate NO production. In the absence of polyphenols, insulin stimulation increased (P<0.05) indices of NO production, phosphorylated to total Akt (p‐AktSer473:Akt), and endothelial nitric oxide synthase (p‐eNOSser1177:eNOS) in cells grown in 5mM but not 25mM glucose. Pretreatment of cells with polyphenol metabolites prior to 25mM glucose exposure preserved insulin stimulated increases (P<0.05) in NO, p‐AktSer473:Akt and p‐eNOSser1177:eNOS. These effects may be secondary to oxidative stress as elevations (P<0.05) in reactive oxygen and nitrogen species in cells treated with 25mM glucose were completely prevented by all polyphenol metabolites. These data indicate that biological metabolites of quercetin, resveratrol, and grape seed extract protected against gluocotoxic impairment of insulin dependent NO bioavailability, and preserved the insulin ‐ Akt ‐ eNOS signaling axis.
We have shown in bovine aortic endothelial cells (BAECs) treated with palmitate (P) and in arteries from obese mice that protein phosphatase 2A (PP2A) associates directly with eNOS. When PP2A co‐localizes with eNOS, interactions among Akt‐Hsp90‐eNOS are disrupted, p‐eNOS/eNOS is decreased, and NO bioavailability is impaired. We hypothesized that PP2A activation is sufficient to disrupt interactions among Akt‐Hsp90‐eNOS in arteries from fat‐fed mice. To test efficacy of the in vivo PP2A inhibitor Lixte Biotechnology 1 (LB1, Setauket, NY), BAECs were treated (3h) ± 500 uM P ± 4 uM LB1. P‐induced increases (p<0.05) in PP2A activity, and reductions (p<0.05) in p‐eNOS/eNOS and NO production (ELISA and EPR), were prevented by LB1. To determine the safety / efficacy of LB1, mice were treated (1 mg/kg/day IP) ± LB1 for 21 days. PP2A activity was reduced (p<0.05) and p‐PP2A Y307 to PP2A was elevated (p<0.05) in arteries from LB1 vs. vehicle‐treated mice after 3 and 21 days. To confirm the importance of PP2A vs. other phosphatases, BAECs incubated ± PP1 (3 uM tautomycin), PP2B (100 nM cyclosporine), or PP2A (4 uM LB1) inhibition. P‐induced reductions (p<0.05) in p‐eNOS/eNOS were prevented only by LB1. Finally, 7‐week old, male C57B16 mice consumed standard (CON, n=20) or high‐fat (HF, n=20) chow for 12‐weeks. Subgroups (n=10) of CON and HF mice received IP injections of saline (vehicle; V) or LB1 for the last 14‐days. HF mice gained weight and developed peripheral glucose intolerance vs. CON mice regardless of LB1 treatment. p‐eNOS/eNOS was impaired (53±6%; p<0.05) in vascular homogenates from HF‐V vs. all groups. After eNOS immunoprecipitation (IP), the association between: (i) PP2A:eNOS increased 95±11%, and the co‐localization between (ii) Akt:eNOS decreased 53±8%, and (iii) Hsp90:eNOS decreased 56±12%, in HF‐V vs. CON mice (all p<0.05). These findings suggest that PP2A activation is sufficient to disrupt interactions among Akt‐Hsp90‐eNOS in arteries from fat‐fed mice. Grant Funding Source: ADA 7‐08‐RA‐164, ADA1‐12‐BS‐208, 2R15HL091493
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