Determination of transdermal sildenafil in nude mouse skin by reversed-phase high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatographic method was developed for the determination of sildenafil transdermal permeation of nude mouse skin. A reversed-phase column with UV detection at 224 nm was used for chromatographic separation. The mobile phase consisted of 32% acetonitrile with 0.2% phosphoric acid in water at pH 5.3 adjusted with 10 M NaOH with the flow-rate set at 1.0 ml/min. The limit of quantitation achieved was 5 ng/ml, and the calibration curve showed good linearity over the concentration range of 5–500 ng/ml. The relative standard deviations of within- and between-day analyses were all within 15%. Sildenafil was found to be stable between pH 3 and 12 during 24-h incubation with skin. After transdermal administration of 15.8 μg/ml of sildenafil to nude mouse skin, it was detected as early as 15 min. The transport amount of sildenafil could be quantitated and, at pH 8–11, had the highest permeation rate in nude mouse skin.     

Internalized Gold Nanoparticles Do Not Affect the Osteogenesis and Apoptosis of MG63 Osteoblast-Like Cells: A Quantitative,In Vitro Study

The long-term toxicity effects of gold nanoparticles (GNPs) on the proliferation and differentiation of a progenitor cell line, MG63 osteoblast-like cells, was investigated. These cells were treated for 20 hours with two media that contained 10 nm GNPs at concentrations of 1 ppm and 10 ppm. The mitosis of the GNP-treated MG63 was observed after at least 21 hours using dark-field and fluorescence microscopy. The TEM, LSCM and dark-field hyperspectral images indicated that the late endosomes in cells that contained aggregated GNPs were caused by vesicle fusion. Subsequently, after 21 days of being cultured in fresh medium, the specific nodule-like phenotypes and bone-associated gene expression of the treated MG63 cells exhibited the same behaviors as those of the control group. Statistically, after 21 days, the viability of the treated cells was identical to that of the untreated ones. During the cell death program analysis, the apoptosis and necrosis percentages of cells treated for 8 or fewer days were also observed to exhibit no significant difference with those of the untreated cells. In summary, our experiments show that the long-term toxicity of GNPs on the osteogenetic differentiation of MG63 is low. In addition, because of their low toxicity and non-biodegradability, GNPs can potentially be used as biomarkers for the long-term optical observation of the differentiation of progenitor or stem cells based on their plasmonic light-scattering properties.     

Prediction and Analysis of Antibody Amyloidogenesis from Sequences

Antibody amyloidogenesis is the aggregation of soluble proteins into amyloid fibrils that is one of major causes of the failures of humanized antibodies. The prediction and prevention of antibody amyloidogenesis are helpful for restoring and enhancing therapeutic effects. Due to a large number of possible germlines, the existing method is not practical to predict sequences of novel germlines, which establishes individual models for each known germline. This study proposes a first automatic and across-germline prediction method (named AbAmyloid) capable of predicting antibody amyloidogenesis from sequences. Since the amyloidogenesis is determined by a whole sequence of an antibody rather than germline-dependent properties such as mutated residues, this study assess three types of germline-independent sequence features (amino acid composition, dipeptide composition and physicochemical properties). AbAmyloid using a Random Forests classifier with dipeptide composition performs well on a data set of 12 germlines. The within- and across-germline prediction accuracies are 83.10% and 83.33% using Jackknife tests, respectively, and the novel-germline prediction accuracy using a leave-one-germline-out test is 72.22%. A thorough analysis of sequence features is conducted to identify informative properties for further providing insights to antibody amyloidogenesis. Some identified informative physicochemical properties are amphiphilicity, hydrophobicity, reverse turn, helical structure, isoelectric point, net charge, mutability, coil, turn, linker, nuclear protein, etc. Additionally, the numbers of ubiquitylation sites in amyloidogenic and non-amyloidogenic antibodies are found to be significantly different. It reveals that antibodies less likely to be ubiquitylated tend to be amyloidogenic. The method AbAmyloid capable of automatically predicting antibody amyloidogenesis of novel germlines is implemented as a publicly available web server at http://iclab.life.nctu.edu.tw/abamyloid.     

A novel prenylated C6–C3 compound with estrogen-like activity from the fruits of Illicium arborescens

A novel C₆–C₃ prenylated compound, illicarborene A (1), together with illioliganfunone D (2), 1-allyl-3,5-dimethoxy-4-(3-methylbut-2-enyloxy)benzene (3), (?)-illicinone A (4), (?)-illicinone B (5) and (?)-illicinone A derivative (6) was isolated and characterized from the fruits of Illicium arborescens Hayata. Compound 1 possesses a new class of tricyclic 6/6/5 ring system. The structure of 1 was determined by spectroscopic analysis such as ¹H–¹H COSY, HMQC, HMBC, and NOESY, and confirmed by chemical reaction to yield 7. Compounds 1–5 were found to increase proliferative activity in primary cell culture of osteoblast cells.     

Spry1 and Spry4 Differentially Regulate Human Aortic Smooth Muscle Cell Phenotype via Akt/FoxO/Myocardin Signaling

Background

Changes in the vascular smooth muscle cell (VSMC) contractile phenotype occur in pathological states such as restenosis and atherosclerosis. Multiple cytokines, signaling through receptor tyrosine kinases (RTK) and PI3K/Akt and MAPK/ERK pathways, regulate these phenotypic transitions. The Spry proteins are feedback modulators of RTK signaling, but their specific roles in VSMC have not been established.

Methodology/Principal Findings

Here, we report for the first time that Spry1, but not Spry4, is required for maintaining the differentiated state of human VSMC in vitro. While Spry1 is a known MAPK/ERK inhibitor in many cell types, we found that Spry1 has little effect on MAPK/ERK signaling but increases and maintains Akt activation in VSMC. Sustained Akt signaling is required for VSMC marker expression in vitro, while ERK signaling negatively modulates Akt activation and VSMC marker gene expression. Spry4, which antagonizes both MAPK/ERK and Akt signaling, suppresses VSMC differentiation marker gene expression. We show using siRNA knockdown and ChIP assays that FoxO3a, a downstream target of PI3K/Akt signaling, represses myocardin promoter activity, and that Spry1 increases, while Spry4 decreases myocardin mRNA levels.

Conclusions

Together, these data indicate that Spry1 and Spry4 have opposing roles in VSMC phenotypic modulation, and Spry1 maintains the VSMC differentiation phenotype in vitro in part through an Akt/FoxO/myocardin pathway.     

Anti-oxidant activity and effect of Pinus morrisonicola Hay. on the survival of leukemia cell line U937

The free radical scavenging and anti-cancer activites of Pinus morrisonicola Hay. were studied using different parts of the pine, namely, needle, bark and cone. Results showed that pine needle water extract has the highest scavenging superoxide anion activity and the lowest IC50 value in inhibiting superoxide anion formation; however, the bark water extract showed the best anti-lipid peroxidation activity. Additionally, needle water extract displayed the highest inhibition of leukemia cell line U937 growth. The results indicated that P. morrisonicola Hay. possesses potential chemopreventative and therapeutic properties.     

Effects of lamivudine on the hepatitis B virus specific CD8+ cytotoxic T lymphocyte responses via peptide-MHC tetrameric complexes assay

Lamivudine, an oral nucleoside analogue, inhibitshepatitis B virus (HBV) replication. It has been shown tobe able to restore T cell responsiveness and to induce atype 1 T helper cell (Th 1) immunity in chronic HBVpatients. To further examine the effects of lamivudineon cytotoxic T lmphocyte (CTL) responses, two HBVantigenic peptide-HLA-A2 tetrameric complexes containingpeptides derived from HBV core protein (residues 18-27;FLPSDFFPSV) and polymerase (residue 551-559; YMDDVVLGA)were constructed. These two tetramers were used toserially determine the frequency of HBV antigen-specificCD8⁺ T cells before and during the treatment oflamivudine. The specificity of these tetramers wasconfirmed by (a) nonstaining of CD8⁺ T cells fromHLA-A2-negative HBV patients, (b) having variablefrequency data in the different teteramer measurement,and (c) showing peptide-specific CTL activity in thesorted tetramer-stanining CD8⁺T cells. Lowfrequency of HBV-specific CTLs was measured for bothtetramers before lamivudine treatment. However, thenumber of CD8⁺ T cells specific for HBV core 18-27increased significantly during lamivudine treatment. Incontrast, relatively lower frequency of HBV pol 551-559specific CD8⁺ T cells was persistently measuredafter lamivudine treatment. These results indicated thatthe lamivudine treatment could enhance HBV specific CTLresponses.