Engineered dityrosine-bonding of the RSV prefusion F protein imparts stability and potency advantages.

Viral fusion proteins facilitate cellular infection by fusing viral and cellular membranes, which involves dramatic transitions from their pre- to postfusion conformations. These proteins are among the most protective viral immunogens, but they are metastable which often makes them intractable as subunit vaccine targets. Adapting a natural enzymatic reaction, we harness the structural rigidity that targeted dityrosine crosslinks impart to covalently stabilize fusion proteins in their native conformations. We show that the prefusion conformation of respiratory syncytial virus fusion protein can be stabilized with two engineered dityrosine crosslinks (DT-preF), markedly improving its stability and shelf-life. Furthermore, it has 11X greater potency as compared with the DS-Cav1 stabilized prefusion F protein in immunogenicity studies and overcomes immunosenescence in mice with simply a high-dose formulation on alum.

Metabolic and molecular responses to leucine-enriched branched chain amino acid supplementation in the skeletal muscle of alcoholic cirrhosis.

UNLABELLED: Skeletal muscle loss (sarcopenia) is a major clinical complication in alcoholic cirrhosis with no effective therapy. Skeletal muscle autophagic proteolysis and myostatin expression (inhibitor of protein synthesis) are increased in cirrhosis and believed to contribute to anabolic resistance. A prospective study was performed to determine the mechanisms of sarcopenia in alcoholic cirrhosis and potential reversal by leucine. In six well-compensated, stable, alcoholic patients with cirrhosis and eight controls, serial vastus lateralis muscle biopsies were obtained before and 7 hours after a single oral branched chain amino acid mixture enriched with leucine (BCAA/LEU). Primed-constant infusion of l-[ring-(2) H5 ]-phenylalanine was used to quantify whole-body protein breakdown and muscle protein fractional synthesis rate using liquid chromatography/mass spectrometry. Muscle expression of myostatin, mammalian target of rapamycin (mTOR) targets, autophagy markers, protein ubiquitination, and the intracellular amino acid deficiency sensor general control of nutrition 2 were quantified by immunoblots and the leucine exchanger (SLC7A5) and glutamine transporter (SLC38A2), by real-time polymerase chain reaction. Following oral administration, plasma BCAA concentrations showed a similar increase in patients with cirrhosis and controls. Skeletal muscle fractional synthesis rate was 9.63 ± 0.36%/hour in controls and 9.05 ± 0.68%/hour in patients with cirrhosis (P = 0.54). Elevated whole-body protein breakdown in patients with cirrhosis was reduced with BCAA/LEU (P = 0.01). Fasting skeletal muscle molecular markers showed increased myostatin expression, impaired mTOR signaling, and increased autophagy in patients with cirrhosis compared to controls (P < 0.01). The BCAA/LEU supplement did not alter myostatin expression, but mTOR signaling, autophagy measures, and general control of nutrition 2 activation were consistently reversed in cirrhotic muscle (P < 0.01). Expression of SLC7A5 was higher in the basal state in patients with cirrhosis than controls (P < 0.05) but increased with BCAA/LEU only in controls (P < 0.001). CONCLUSIONS: Impaired mTOR1 signaling and increased autophagy in skeletal muscle of patients with alcoholic cirrhosis is acutely reversed by BCAA/LEU.

Partially hydrolyzed soy protein shows enhanced transport of amino acids compared to nonhydrolyzed protein across an intestinal epithelial cell monolayer.

Consumption of protein hydrolysates has been proposed to stimulate muscle anabolism more than intact (nonhydrolyzed) proteins via accelerated delivery of amino acids for muscle protein synthesis (MPS). We evaluated whether the rate of amino acid uptake and transport across intestinal cells was enhanced for soy protein hydrolysates versus nonhydrolyzed soy protein. Intact and partially hydrolyzed proteins were subject to simulated gut digestion and applied to the apical surface of Caco-2 monolayers. Basolateral media was harvested after 3 h and quantitatively analyzed for free amino acids using ion-exchange chromatography and comparison to an included reference standard. Basolateral concentrations of all amino acids were higher (mean 32%) for hydrolyzed versus nonhydrolyzed protein with the greatest differences in histidine, lysine, and valine. Scale-up production of the soy protein hydrolysate did not diminish its enhanced absorption properties. These data support the hypothesis that hydrolyzed soy protein may provide dietary amino acids that are more rapidly transported across the intestinal epithelium versus intact soy protein. This would be important under conditions where rapid and increased levels of amino acids are needed such as in the stimulation of MPS.

Fatty acid, amino acid, mineral and antioxidant contents of acha (Digitaria exilis) grown on the Jos Plateau, Nigeria.

Digitaria exilis (Kippist) Stapf (also known as acha, hungry rice) has been cultivated for millennia in the dry savannahs of West Africa, but much remains to be learned about its nutritional properties. Acha was collected in four villages in Northern Nigeria and analyzed for fatty acids, minerals, amino acids and antioxidant content. Fatty acids accounted for 1.91% of the dry weight, with 47.4% linoleic acid and 30.5% oleic acid. The content of the essential minerals, copper, magnesium, molybdenum, zinc and calcium averaged 4.88, 1060, 0.23, 23.0 and 172 µg/g, respectively. The protein content was 6.53% and the essential amino acid pattern, except for lysine, compared favorably to a World Health Organization (WHO) reference protein. The total polyphenolic content of methanolic extracts of acha matched that of common cereals (for example, maize, rice, wheat) and the extracts contained substantial amounts of free-radical scavenging substances. Thus, acha is a source of many nutrients critical to human health.

Adenosine 5'-phosphosulfate reductase (APR2) mutation in Arabidopsis implicates glutathione deficiency in selenate toxicity.

APR2 is the dominant APR (adenosine 5'-phosphosulfate reductase) in the model plant Arabidopsis thaliana, and converts activated sulfate to sulfite, a key reaction in the sulfate reduction pathway. To determine whether APR2 has a role in selenium tolerance and metabolism, a mutant Arabidopsis line (apr2-1) was studied. apr2-1 plants had decreased selenate tolerance and photosynthetic efficiency. Sulfur metabolism was perturbed in apr2-1 plants grown on selenate, as observed by an increase in total sulfur and sulfate, and a 2-fold decrease in glutathione concentration. The altered sulfur metabolism in apr2-1 grown on selenate did not reflect typical sulfate starvation, as cysteine and methionine levels were increased. Knockout of APR2 also increased the accumulation of total selenium and selenate. However, the accumulation of selenite and selenium incorporation in protein was lower in apr2-1 mutants. Decreased incorporation of selenium in protein is typically associated with increased selenium tolerance in plants. However, because the apr2-1 mutant exhibited decreased tolerance to selenate, we propose that selenium toxicity can also be caused by selenate's disruption of glutathione biosynthesis leading to enhanced levels of damaging ROS (reactive oxygen species).

Endothelial inflammation correlates with subject triglycerides and waist size after a high-fat meal.

A rise in postprandial serum triglycerides (PP-sTG) can potentiate inflammatory responses in vascular endothelial cells (ECs) and thus serves as an independent risk factor for predicting increased cardiovascular morbidity. We examined postprandial triglyceride-rich lipoproteins (PP-TGRLs) in subjects ranging from normal to hypertriglyceridemic for their capacity to alter EC acute inflammatory responses. Cultured human aortic ECs (HAECs) were conditioned with PP-TGRLs isolated from human serum at the peak after a moderately high-fat meal. VLDL particle size increased postprandially and varied directly with the subject's PP-sTG level and waist circumference. PP-TGRL particles bound to HAECs and were internalized via LDL receptor-mediated endocytosis. PP-TGRL alone did not induce an inflammatory response over the range of individuals studied. However, combined with low-dose TNF-α stimulation (0.3 ng/ml), it elicited a net 10-15% increase above cytokine alone in the membrane expression of VCAM-1, ICAM-1, and E-selectin, which was not observed with fasting TGRLs. In contrast to upregulation of ICAM-1 and E-selectin, VCAM-1 transcription and expression varied in direct proportion with individual PP-sTG and waist circumference. The extent of monocyte arrest on inflamed HAECs under shear stress also correlated closely with VCAM-1 expression induced by conditioning with PP-TGRL and TNF-α stimulation. This ex vivo approach provides a quantitative means to assess an individual's inflammatory potential, revealing a greater propensity for endothelial inflammation in hypertriglyceridemic individuals with abdominal obesity.

Changes on the physiological lactonase activity of serum paraoxonase 1 by a diet intervention for weight loss in healthy overweight and obese women.

Low caloric diet (LCD) is used for weight loss. Paraoxonase 1 (PON-1) is associated with the antioxidant functions of high-density lipoprotein (HDL). Among limited data on the relationships between obesity and PON-1, there has been no study on the effects of a stand-alone LCD on the physiological lactonase activity of PON-1. We investigated the prospective effects of LCD intervention (2 months) for weight loss on serum PON-1 activities (lactonase, arylesterase [mono-esterase] and tri-esterase) and HDL cholesterol (HDL-C), and their association with low-density lipoprotein cholesterol (LDL-C) in overweight and non-morbidly obese but otherwise healthy women (n = 30; mean age, 50.3 years; mean body mass index [BMI], 28.5 kg/m(2)). In addition to the data such as BMI, blood pressure, blood glucose and lipids, PON-1 activities were examined between pre- and post-intervention. The intervention reduced all metabolic outcomes, and PON-1 lactonase activity (determined with 5-[thiobutyl]butyrolactone) significantly decreased by 6.1%, paralleled by arylesterase (by 7.3%) and tri-esterase (by 7.8%). In multiple regression analysis, the percent change of PON-1 lactonase was significantly, positively and independently correlated to that of LDL-C (beta = 0.51), HDL-C (beta = 0.40), and BMI (beta = 0.37). Our results showed that the solo diet treatment on weight loss might reduce serum PON-1 lactonase activity with reduced HDL-C and LDL-C. The relationship between the lactonase and LDL-C may be adaptive, plausibly hypothesizing less need for PON-1 activity as an antioxidant property to protect lipoproteins. Further research is needed to confirm this prediction.

Extracellular potassium dependency of block of HERG by quinidine and cisapride is primarily determined by the permeant ion and not by inactivation.

Drug induced Long QT syndrome results primarily from block of the cardiac potassium channel HERG (human-ether-a-go-go related gene). In some cases prolongation of the QT interval can result in the lethal arrhythmia torsade de pointes, an arrhythmia characterized by a rapid heart rate and severely compromised cardiac output. Many patients requiring medication present with abnormal serum electrolyte levels due to a variety of conditions including gastrointestinal dysfunction, renal and endocrine disorders, diuretic use, alcoholism and aging. Extracellular cations have significant influence on HERG channel gating and in some instances they have been shown to alter drug block of HERG. However, the mechanisms by which drug block is altered in different extracellular cation solutions are not well understood. In this study, HERG block by quinidine and cisapride was assessed in extracellular solutions of calcium, potassium, rubidium, cesium and tetraethylammonium (TEA) using two-electrode voltage clamping of Xenopus oocytes. Consistent with previous reports we show that increases in extracellular potassium reduce HERG block by quinidine and cisapride. We also show that increasing extracellular rubidium and cesium reduced HERG block by quinidine and cisapride whereas increasing extracellular calcium and extracellular TEA did not alter HERG block by quinidine and cisapride. These results demonstrate that at lower extracellular potassium concentrations, the permeant ion is almost exclusively responsible for the reduction in quinidine and cisapride block of HERG due to increases in extracellular potassium.

Caffeic and chlorogenic acids in Ilex paraguariensis extracts are the main inhibitors of AGE generation by methylglyoxal in model proteins.

The present study concentrates on the evaluation of the anti-glycation effect of some bioactive substances present in yerba maté (Ilex paraguariensis): 5-caffeoylquinic acid, caffeic acid and a sapogenin (oleanolic acid). Bovine serum albumin and histones were incubated in the presence of methylglyoxal with or without the addition of 5-caffeoylquinic acid, caffeic acid and oleanolic acid. After the incubation period, advanced glycation end product (AGE) fluorescence spectra were performed and protein structural changes were evaluated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. Chlorogenic acid, caffeic acid are the main substances responsible for the anti-glycation effect of maté tea.

Protective action of Ilex paraguariensis extract against free radical inactivation of paraoxonase-1 in high-density lipoprotein.

Paraoxonase 1 (PON-1), an antioxidant enzyme carried mainly by HDL, has been shown to display cardioprotective effects. In this study, we investigated whether the polyphenol-rich beverage mate, obtained from extract of Ilex paraguariensis (IP), prevented the loss of PON-1 activity from HDL during oxidant stress. The peroxide radical generator 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) induced time- and dose-dependent oxidation of HDL, as measured by lipoperoxide content, which is accompanied by a parallel decrease in the activity of PON-1 (p<0.001). IP extract (2-20 microL/mL) afforded time- and concentration-dependent inhibition of the oxidation, with preservation of apoA-I structure and PON-1 activity. Healthy volunteers drank either 0.5 L of IP extract, 0.5 L of coffee and milk or nothing. PON-1 activity increased an average of 10% above the changes seen when the intake was coffee and milk (p<0.05). In conclusion, we demonstrate that IP extract may, to some extent, prevent the loss of the antiatherogenic function of HDL afforded by PON-1 when the particle is under oxidant stress.

Acrolein inactivates paraoxonase 1: changes in free acrolein levels after hemodialysis correlate with increases in paraoxonase 1 activity in chronic renal failure patients.

BACKGROUND: Acrolein is a very reactive aldehyde present in cigarette smoke and endogenously generated by pathways such as lipid peroxidation and threonine metabolism by phagocytes. Acrolein has been shown to affect uptake of cholesterol by HDL. We hypothesized that acrolein could also have deleterious effects on paraoxonase 1 (PON-1) activity. We also determined whether free serum acrolein levels are higher in renal failure, and assessed whether they decrease after hemodialysis (HD) and whether this change correlates with increases in PON-1 activity. METHODS: We incubated human HDL with 0-10 mmol/l acrolein for 2 h and measured PON-1 activity and structural changes. Acrolein was also measured in 40 end stage renal disease (ESRD) patients (before and after a hemodialysis session), and 40 control subjects. RESULTS: We found that acrolein inhibits PON-1 activity in HDL in a time and concentration dependent fashion. Inhibition occurred at 40% at 0.5 mmol/l and was cancelled by cysteine but not by aminoguanidine or carnosine. We confirm that free serum acrolein levels are higher in chronic renal failure patients and demonstrate that they are partially removed by HD. Decrease in acrolein levels after dialysis correlate with increases in PON-1 activity (r=0.32, p 0.01). CONCLUSION: Acrolein inactivates paraoxonase 1 in HDL, a process that is inhibited by N-acetylcysteine. We confirm that acrolein levels are higher in ESRD and show for the first time, data supporting that acrolein is partially removed by hemodialysis. Decrease in acrolein levels after dialysis correlates with increase in PON-1 activity. This could offer new insights to explain low PON-1 activities in smokers and renal failure subjects as well as pointing at thiol-conserving reducing compounds such as N-acetylcysteine, as putative therapeutic palliatives.

Paraoxonase-1 concentrations in end-stage renal disease patients increase after hemodialysis: correlation with low molecular AGE adduct clearance.

BACKGROUND: Hemorrhagic stroke and ischemic heart disease continue to be key problems in patients with end stage renal failure. Reduced serum paraoxonase (PON-1) activity has been described in these patients, which could contribute to the accelerated development of atherosclerosis. We hypothesized that retention of uremic toxins and or "middle molecules" including advanced glycation (AGE) free adducts and peptides could play a mechanistic role in decreasing PON-1 activity. METHODS: We enrolled 22 ESRD patients undergoing hemodialysis in whom paired pre- and post-dialysis samples were studied along with 30 age-matched control subjects. RESULTS: ESRD patients showed a 76% decrease in PON-1 activity. As expected, ESRD patients had an increase in lipoperoxides and advanced oxidation protein products (AOPP). Our patients had a 3-fold increase in serum AGEs and a striking 10-fold increase in low molecular weight (<10 kDa) AGEs. Post-dialysis samples in all patients displayed an increase in PON-1 activity, which ranged from 4 to 40% of the predialysis value. HDL-cholesterol, apoAI, free cholesterol (as a LCAT surrogate), HDL-subclasses and TG did not change significantly after dialysis. Changes in PON-1 activity display a good correlation (r=0.66, p<0.001) with rates in which creatinine and urea are cleared. Clearance of low molecular weight AGEs after hemodialysis explains 79% of the changes in PON-1 activity and are hence a much better predictor than creatinine changes (r=0.89, p<0.00). In vitro incubation of paraoxonase with serum ultrafiltrates show a time and concentration dependent inhibition of PON-1 by the ultrafiltrates, an inhibition that is up to 3 times higher (from 8 to 24%) when chronic renal failure patients are the source of the ultrafiltrate. CONCLUSION: We showed that HD results in a significant, consistent increase in the activity of the antioxidant enzyme PON-1. The effect, correlates with the effectiveness of dialysis to clear creatinine and urea, and with the clearance of AGE adducts of low molecular weight. This effect was replicated in vitro, showing time and dose dependency. Our results suggest that another cause for the observed lower PON-1 concentrations in CRF are the retention of low-middle molecules and demonstrate a positive effect of hemodialysis in the delicate oxidant-antioxidant state of these patients, that should be weighted against other pro-oxidant effects that have also been shown to occur previously. If the hypothesis that AGEs are the main culprits is proved in further research, this opens a putative therapeutic avenue for AGE blockers in ESRD.

Nitration of human plasminogen by RAW 264.7 macrophages reduces streptokinase-induced plasmin activity.

Despite many years of study, clinical trials of new drugs to prevent thrombosis have often been disappointing. Part of the problem lies in our incomplete understanding of the regulation of plasminogen activation and/or inhibition in vivo. We have previously shown that in vitro nitration of plasminogen in plasma by peroxynitrite resulted in decreased plasmin activity. We hypothesized that macrophages may be agents of plasminogen nitration and designed this study to prove this hypothesis. We first better characterized our previous observations using purified plasminogen instead of whole plasma, studied the time and concentration dependence of these reactions, and co-incubated plasminogen with macrophages, as well as with non-inflammatory cells as controls, to assess nitration and impaired activity. When plasminogen (10 micromol/L) is incubated in the presence of SIN-1 (0.01-2 mmol/L), plasmin activity (generated by streptokinase) is reduced in a time- and concentration-dependent fashion. We performed experiments incubating human plasminogen in the presence of murine RAW264.7 macrophages, allowing for free diffusion of reactive oxygen species, while preventing the action of proteases. In this way we show that incubation of plasminogen with macrophages also decreases plasmin activity, while increasing nitration of the molecule, an effect that is already apparent after 2 h and reaches a plateau of 60% inhibition after 24 h of incubation. This effect appears specific for macrophages, since 31EG4 murine mammary cells used in parallel and under the same conditions failed to produce any deleterious changes in plasminogen. Our data on quick functional inactivation of plasminogen by nitration, mediated by macrophages, adds a new pathophysiological dimension to our previous work showing plasminogen as a target for peroxynitrite damage. Nitrosative stress may be implicated in impaired fibrinolysis. New therapeutic approaches for nitrosative stress in atherosclerosis and diabetes should limit the formation of superoxides and peroxynitrite.