Auto-sumoylation of the Ubc9 E2 SUMO-conjugating Enzyme Extends Cellular Lifespan.

Calorie restriction (CR) provides anti-aging benefits through diverse processes, such as reduced metabolism and growth and increased mitochondrial activity. Although controversy still exists regarding CR-mediated lifespan effects, many researchers are seeking interventions that mimic the effects of CR. Yeast has proven to be a useful model system for aging studies, including CR effects. We report here that yeast adapted through in vitro evolution to the severe cellular stress caused by loss of the Ulp2 SUMO-specific protease exhibit both enhanced growth rates and replicative lifespan, and they have altered gene expression profiles similar to those observed in CR. Notably, in certain evolved ulp2Δ lines, a dramatic increase in the auto-sumoylation of Ubc9 E2 SUMO-conjugating enzyme results in altered regulation of multiple targets involved in energy metabolism and translation at both transcriptional and post-translational levels. This increase is essential for the survival of aged cells and CR-mediated lifespan extension. Thus, we suggest that high Ubc9 auto-sumoylation exerts potent anti-aging effects by promoting efficient energy metabolism-driven improvements in cell replication abilities. This potential could be therapeutically explored for the development of novel CR-mimetic strategies.

Seed-assembly-mediated fabrication and application of highly branched gold nanoshells having hollow and porous morphologies.

Highly branched gold nanoshells (BAuNSs) having hollow and porous morphologies have been fabricated via a seed-assembly-mediated strategy. Gold seed assemblies can be prepared by removal of SiO2nanotemplates with help of polyvinylpyrrolidone (PVP) molecules, which weakly link gold nanoparticles together even after SiO2etching. L-3,4-dihydroxy phenylalanine (L-DOPA) and AgNO3are employed as shape-directing agents to induce the anisotropic growth of gold. BAuNSs exhibit 7.4 and 4.4 times stronger activities than SiO2@Au nanoparticles in catalysis and surface-enhanced Raman scattering (SERS) applications, respectively, due to their large surface areas and numerous hot spots. It is necessary to find the optimal amount of gold deposition in fabrication to effectively utilize the hollow and porous morpologies of BAuNSs for catalysis and SERS applications. Overgrown nanobranches can fill the nanopores and nanogaps of BAuNSs, resulting in decrease of activities in applications. Overall, the seed-assembly-mediated fabrciation can be employed to produce plasmonic nanostructures having unique morphologies and high application activities.

Photothermal structural modification of porous gold nanoshells via pulsed-laser irradiation: effects of laser wavelengths and surface conditions.

Pulsed lasers are promising candidates for fabricating plasmonic nanoparticles having unique structures, and there is a strong need for studies on the detailed effects of various experimental conditions on laser-induced fabrications. In this work, we demonstrate the effects of laser wavelengths and nanoparticle surface conditions, as well as laser fluences, in the structural modification of porous gold nanoshells induced by picosecond pulse irradiation. Laser wavelengths play a critical role in the modification because irradiating laser pulses excite not only porous gold nanoshells but also gold nanospheres, which have been produced via the melting of irradiated porous gold nanoshells. Significantly different localized surface plasmon resonances of gold nanospheres and porous gold nanoshells make the effect of laser wavelengths noticeable. The polyvinylpyrrolidone (PVP) concentration of colloid containing porous gold nanoshells also modifies the deformation of the structures. Gold nanostructures become positively charged by the irradiation, strengthening gold-PVP attraction. The stronger binding affinity of PVP is considered to reduce the deformation of irradiated porous gold nanoshells.

Boosting Visible-Light Photocatalytic Redox Reaction by Charge Separation in SnO2 /ZnSe(N2 H4 )0.5 Heterojunction Nanocatalysts.

In this work, environmentally friendly photocatalysts with attractive catalytic properties are reported that have been prepared by introducing SnO2 quantum dots (QDs) directly onto ZnSe(N2 H4 )0.5 substrates to induce advantageous charge separation. The SnO2 /ZnSe(N2 H4 )0.5 nanocomposites could be easily synthesized through a one-pot hydrothermal process. Owing to the absence of capping ligands, the attached SnO2 QDs displayed superior photocatalytic properties, generating many exposed reactive surfaces. Moreover, the addition of a specified amount of SnO2 boosted the visible-light photocatalytic activity; however, the presence of excess SnO2 QDs in the substrate resulted in aggregation and deteriorated the performance. The spectroscopic data revealed that the SnO2 QDs act as a photocatalytic mediator and enhance the charge separation within the type II band alignment system of the SnO2 /ZnSe(N2 H4 )0.5 heterojunction photocatalysts. The separated charges in the heterojunction nanocomposites promote radical generation and react with pollutants, resulting in enhanced photocatalytic performance.

Myeloid deletion of SIRT1 aggravates serum transfer arthritis in mice via nuclear factor-κB activation.

OBJECTIVE: SIRT1 modulates the acetylation of the p65 subunit of nuclear factor-κB (NF-κB) and plays a pivotal role in the inflammatory response. This study sought to assess the role of SIRT1 in rheumatoid arthritis (RA) using a myeloid cell-specific SIRT1 knockout (mSIRT1 KO) mouse. METHODS: mSIRT1 KO mice were generated using the loxP/Cre recombinase system. K/BxN serum transfer arthritis was induced in mSIRT1 KO mice and age-matched littermate loxP control mice. Arthritis severity was assessed by clinical and pathological scoring. The levels of inflammatory cytokines in the serum and joints were measured by ELISA. Migration, M1 polarization, cytokine production, osteoclastogenesis, and p65 acetylation were assessed in bone marrow-derived monocytes/macrophages (BMMs). RESULTS: mSIRT1 KO mice showed more severe inflammatory arthritis and aggravated pathological findings than control mice. These effects were paralleled by increases in IL-1, TNF-α, TRAP-positive osteoclasts, and F4/80⁺ macrophages in the ankles of mSIRT1 KO mice. In addition, BMMs from mSIRT1 KO mice displayed hyperacetylated p65 and increased NF-κB binding activity when compared to control mice, which resulted in increased M1 polarization, migration, pro-inflammatory cytokine production, and osteoclastogenesis. CONCLUSION: Our study provides in vivo evidence that myeloid cell-specific deletion of SIRT1 exacerbates inflammatory arthritis via the hyperactivation of NF-κB signaling, which suggests that SIRT1 activation may be beneficial in the treatment of inflammatory arthritis.

Interspecies scaling of oleanolic acid in mice, rats, rabbits and dogs and prediction of human pharmacokinetics.

This study was conducted to predict the pharmacokinetics of oleanolic acid in humans based on animal data by allometry and several species-invariant time methods. Oleanolic acid was injected intravenously to mice, rats, rabbit and dogs (dose 1 mg/kg). The serum concentration-time profiles of oleanolic acid were best described by bi-exponential equation in all animal species. The average Cl, V ( ss ) and t ( 1/2 ) were 0.065 L/h, 0.019 L and 28.7 min in mice, 0.47 +/- 0.06 L/h, 0.117 +/- 0.029 L and 29.7 +/- 12.2 min in rats, 2.77 +/- 0.88 L/h, 1.83 +/- 0.60 L and 84.4 +/- 16.9 min in rabbits and 14.0 +/- 0.7 L/h, 9.2 +/- 10.1 L and 54.5 +/- 57.2 min in dogs, respectively. Based on animal data, human pharmacokinetic parameters of Cl, V ( ss ) and t (1/2) were predicted by simple allometry. In addition, actual concentration-time profiles obtained from animals were transformed to human profiles by species-invariant times of kallynochron, apolysichron and dienetichron. The predicted human pharmacokinetic parameters of Cl, V ( ss ) and t (1/2) by using simple allometry and species-invariant time transformation method ranged from 48.3-97.2 L/h, 49.1-92.9 L and 45.6-187.2 min, respectively. Those predicted parameters of oleanolic acid may be useful in designing dosing schedules of oleanolic acid in future clinical studies.

Pharmacokinetics of baicalein, baicalin and wogonin after oral administration of a standardized extract of Scutellaria baicalensis, PF-2405 in rats.

The pharmacokinetics of active components such as baicalein, wogonin and oroxylin A were evaluated after oral administration of a purified extract of Scutellaria baicalensis GEORGI (PF-2405) containing the high contents of baicalein, wogonin and oroxylin A to rats. Following oral administration of PF-2405 at 10, 20 and 40 mg/kg dose (equivalent to 4.5, 9.0 and 18 mg/kg baicalein), a major constituent baicalein and its active metabolite baicalin showed dose-linear pharmacokinetics as evidenced by unaltered dose-normalized AUC, dose-normalized Cmax, Ae(0-30h) and GI(30h) values. Following oral administration of PF-2405 at three doses (equivalent to 0.4, 0.8 and 1.6 mg/kg wogonin), dose-normalized Cmax and dose-normalized AUC were comparable between the 20 and 40 mg/kg PF2405 doses, but plasma concentrations of wogonin at 10 mg/kg of PF-2405 were not measurable as they were below limit of quantitation (LOQ; 18 pmol/mL). Following oral administration of PF-2405 at the three doses (equivalent to 1.5, 3.0 and 6.0 mg/kg oroxylin A), the concentrations of oroxylin A in plasma, urine and gastrointestine samples were below the assay LOQ (18 pmol/mL). Significant differences in AUCs, Ae(0-30h) and GI(30h) values for baicalein and baicalin were observed after oral administration of pure baicalein (18 mg/kg) and PF-2405 (40 mg/kg). The increases in AUCs of baicalein and baicalin after oral administration of PF-2405 may have been due to the significant decrease in GO(30h) values for baicalein.

Analysis of carvedilol in human plasma using hydrophilic interaction liquid chromatography with tandem mass spectrometry.

A rapid, sensitive and selective method for the determination of carvedilol in human plasma was developed using hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS). Carvedilol and cisapride (internal standard) were extracted from human plasma with methyl tert-butyl ether at basic pH and analyzed on an Atlantis HILIC Silica column with the mobile phase of acetonitrile-ammonium formate (50 mM, pH 4.5) (90:10, v/v). The analytes were detected using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curve was linear (r=0.9998) over the concentration range of 0.1-200 ng/ml. The lower limit of quantification for carvedilol was 0.1 ng/ml using 50 microl plasma sample. The coefficient of variation and relative error for intra- and inter-assay at four QC levels were 1.6-4.5% and -6.4 to 4.8%, respectively. The absolute and relative matrix effect for carvedilol and cisapride were practically absent. The extraction recoveries of carvedilol and cisapride were 81.6 and 85.2%, respectively. This method was successfully applied to the bioequivalence study of carvedilol in humans.

Dose-linear pharmacokinetics of oleanolic acid after intravenous and oral administration in rats.

The pharmacokinetics of oleanolic acid was evaluated in vitro and in vivo. From Caco-2 cell permeation studies, oleanolic acid was a low permeability compound with no directional effects, suggesting a low in vivo absorption mediated by a passive diffusion. Oleanolic acid was metabolically unstable following incubation with rat liver microsomes in the presence of NADPH. After intravenous injection at doses of 0.5, 1 and 2 mg/kg doses, oleanolic acid showed dose-linear pharmacokinetics as evidenced by unaltered CL (28.6-33.0 ml/min/kg), Vss (437-583 ml/kg), dose-normalized AUC (16.0-17.9 microg min/ml based on 1 mg/kg) and t1/2 (41.9-52.7 min). Following oral administration of oleanolic acid at doses of 10, 25 and 50 mg/kg, Tmax, t1/2, dose-normalized Cmax (66-74 ng/ml based on 25 mg/kg) and dose-normalized AUC (5.4-5.9 microg min/ml based on 25 mg/kg) were comparable between 25 and 50 mg/kg dose, but the plasma concentrations at 10 mg/kg dose were not measurable as they were below the limit of quantitation (2 ng/ml). The absolute oral bioavailability was 0.7% for oral doses of 25 and 50 mg/kg. The extent of urinary excretion was minimal for both i.v. and oral doses. The very low oral bioavailability of oleanolic acid could be due to a poor absorption and extensive metabolic clearance.

Liquid chromatography with tandem mass spectrometry for the simultaneous determination of baicalein, baicalin, oroxylin A and wogonin in rat plasma.

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of baicalein, baicalin, oroxylin A and wogonin, Scutellaria baicalensis active components in rat plasma was developed. After liquid-liquid extraction with 2-(3,4-dimethoxy-phenyl)-5,7-dihydroxy-chromen-4-one as internal standard, baicalein, baicalin, oroxylin A and wogonin were eluted from an Atlantis C(18) column within 7 min with isocratic mobile phase consisting of methanol and 0.1% formic acid (60:40, v/v). The analytes were detected using an electrospray ionization tandem mass spectrometry in the multiple reaction monitoring (MRM) mode. The standard curves were linear (r=1.000) over the concentration ranges of 5-500 ng/ml for baicalein, wogonin and oroxylin A and 5-5000 ng/ml for baicalin. The coefficients of variation and relative errors of baicalein, wogonin, oroxylin A and baicalin for intra- and inter-assay at three or four quality control (QC) levels were 0.8-6.1% and -4.0 to 5.8%, respectively. The lower limits of quantification for baicalein, wogonin, oroxylin A and baicalin were 5ng/ml using 50 microl of plasma sample. This method was successfully applied to the pharmacokinetic study of baicalein, baicalin, wogonin and oroxylin A after an intravenous administration of Scutellariae radix extract to male Sprague-Dawley rats.

Determination of gabapentin in human plasma using hydrophilic interaction liquid chromatography with tandem mass spectrometry.

A rapid, sensitive and selective method for the determination of gabapentin in human plasma was developed using hydrophilic interaction liquid chromatography/tandem mass spectrometry (HILIC/MS/MS). The devised method involved protein precipitation with acetonitrile followed by separation on an Atlantis HILIC silica column using an acetonitrile/ammonium formate mobile phase (100 mM, pH 3.0) (85:15, v/v). Analytes were detected using an electrospray ionization mass spectrometer in the multiple-reaction monitoring mode. The standard curve was linear (r = 1.000) over the concentration range of 50.0-10000 ng/mL. The lower limit of quantification for gabapentin was 50.0 ng/mL (ca. 20 pg gabapentin) using a 10-microL plasma sample. The coefficients of variation and relative errors for intra- and inter-assay at four QC levels (i.e., 50.0, 125, 750, and 7500 ng/mL) were 4.7 to 9.4% and -4.1 to 1.6%, respectively. Absolute and relative matrix effects for gabapentin and metformin were practically absent. Gabapentin and metformin recoveries were 98.5% and 99.0%, respectively. This method was successfully applied to a bioequivalence study of gabapentin in humans.

Hydrophilic interaction liquid chromatography-tandem mass spectrometry for the determination of levofloxacin in human plasma.

A rapid, sensitive and selective hydrophilic interaction liquid chromatography-tandem mass spectrometric (HILIC-MS/MS) method for the determination of levofloxacin in human plasma was developed. Levofloxacin and ciprofloxacin (internal standard) were extracted from human plasma with dichloromethane and analyzed on an Atlantis HILIC Silica column with the mobile phase of acetonitrile-ammonium formate (100 mM, pH 6.5) (82:18 v/v). The analytes were detected using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curve was linear (r>0.999) over the concentration range of 10.0-5000 ng/ml. The lower limit of quantification for levofloxacin was 10.0 ng/ml using 20 microl plasma sample. The coefficient of variation and relative error for intra- and inter-assay at four QC levels were 2.9-7.8% and -7.3% to -2.2%, respectively. The recoveries of levofloxacin and ciprofloxacin were 55.2% and 77.3%, respectively. This method was successfully applied to the pharmacokinetic study of levofloxacin in humans.

Simultaneous determination of piroxicam, meloxicam and tenoxicam in human plasma by liquid chromatography with tandem mass spectrometry.

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of piroxicam, meloxicam and tenoxicam in human plasma was developed. Piroxicam, meloxicam, tenoxicam and isoxicam (internal standard) were extracted from human plasma with ethyl acetate at acidic pH and analyzed on a Sunfire column with the mobile phase of methanol:ammonium formate (15 mM, pH 3.0) (60:40, v/v). The analytes were detected using a mass spectrometer, equipped with electrospray ion source. The instrument was set in the multiple-reaction-monitoring (MRM) mode. The standard curve was linear (r=1.000) over the concentration range of 0.50-200 ng/ml. The coefficient of variation (CV) and relative error (RE) for intra- and inter-assay statistics at three QC levels were 1.0-5.4% and -5.9 to 2.8%, respectively. The recoveries of piroxicam, meloxicam and tenoxicam ranged from 78.3 to 87.1%, with that of isoxicam being 59.7%. The lower limit of quantification for piroxicam, meloxicam and tenoxicam was 0.50 ng/ml using a 100 microl plasma sample. This method was successfully applied to a pharmacokinetic study of piroxicam after application of transdermal piroxicam patches to humans.