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Abstract Genotyping of gDNA rs12041331 (PEAR1), rs6065 (GP1BA), and rs730012 (LTC4S) can provide systematic guidance on the use of aspirin. However, an accurate, reliable and economical approach to simultaneous detection of the above single nucleotide polymorphisms (SNPs) is not reported. Herein, we designed and substantiated an allele-specific (AS) forward primer-superposed amplification analysis for measurement of the SNPs in PEAR1, GP1BA and LTC4S genes, in which the values of ∆Cq (differences in threshold cycles between the wild-type forward primer-based assay and the mutated-type forward primer-based assay) were employed to decide genotype. Mismatch AS forward primers were screened with the singleplex amplification analysis. Moreover, Cq extension optimized by AS forward primer superposition was observed in the selected forward primer-based triplex analysis. Further, robustness assessment of the triplex analysis showed the amplification efficiency ranging from 0.9 to 1.1. Precision test demonstrated the coefficient of variation of less than 2%. And the detective results of 189 DNA samples was completely concordant with that of commercial Sanger sequencing. In summary, we developed a simple, accurate and economical approach to genotyping of rs12041331 (PEAR1), rs6065 (GP1BA) and rs730012 (LTC4S) to provide a valuable pharmacogenomics tool for guidance of aspirin delivery.

Upon binding agonist, the epidermal growth factor receptor (EGFR) is dimerized and auto-phosphorylated to activate downstream pathway that induces diverse physiology and pathology processes. Conventional methods for evaluation of EGFR inhibitors are limited. This study describes a duplexed on-microbead binding assay allowing competitive EGFR inhibitors to be quantificationally evaluated in vitro. Polystyrene microbeads barcoded by fluoresceine isothiocyanate fluorescence as high brightness and low brightness microspheres were coated with receptor tyrosine kinase (RTK) ligand-epidermal growth factor (EGF)/stem cell factor (SCF) and ATP/GTP, respectively. High and low brightness microbeads were mixed and incubated with EGFR and its competitive inhibitor in binding assay buffer. Phycoerythrin (PE) fluorescence-labelled antibody was employed to report the level of EGFR binding to EGF/SCF and ATP/GTP. Values were numbered via PE molecules assessed by quantitative flow cytometry. Results from this study demonstrated that incubation with EGFR identified by PE-labelled antibody can make EGF- and ATP-coated microbeads luminous. And EGF or ATP-competitive EGFR inhibitors, respectively, alleviated this in a concentration-dependent manner. Coating microbeads with SCF or GTP as a negative control cannot capture EGFR. The duplexed on-microbead binding assay in this study might be useful for discovering ligand- and ATP-competitive EGFR inhibitors in a rapid and quantificational approach. [ABSTRACT FROM AUTHOR]

Geniposide is an iridoid glycoside isolated from the fruit of Gardenia jasminoides Ellis used as a Chinese traditional medicine for treatment of generalized vitiligo. Stem cell factor from keratinocyte recognizes and activates its receptor c-kit carried by melanocyte to potent enhance melanocytic melanogenesis that can be suppressed by norepinephrine. This study addresses the action and mechanism of geniposide enhancing melanogenesis in norepinephrine-exposed normal human epidermal melanocyte. Flow cytometry results from this study exhibited the augmentation effect of geniposide on production of c-kit receptor by norepinephrine-exposed normal human epidermal melanocyte. However, geniposide did not affect the production of stem cell factor by norepinephrine-exposed normal human epidermal keratinocyte assessed by cellular enzyme-linked immunosorbent assay (ELISA). ELISA indicated that at the presence of stem cell factor, geniposide was capable of elevating the level of extracellular signal-regulated kinase 1/2 phosphorylation within norepinephrine-exposed normal human epidermal melanocyte, which is known to be involved in stem cell factor/c-kit downstream signalling. And inhibition of c-kit with inhibitory antibody K44.2 completely blocked the increase in geniposide-stimulated extracellular signal-regulated kinase 1/2 phosphorylation. In addition, spectrophotometry demonstrated the enhancement effect of geniposide on melanogenesis (tyrosinase activity and melanin production) in norepinephrine-exposed normal human epidermal melanocyte at the presence of stem cell factor, which was blocked by c-kit inhibitory antibody K44.2. Data from this study suggest that geniposide can enhance melanogenesis by stem cell factor/c-kit signalling in which the expression of c-kit receptor is augmented in norepinephrine-exposed normal human epidermal melanocyte. [ABSTRACT FROM AUTHOR]

Abstract: Geniposide (GP) as an agonist of glucagon-like peptide-1 receptor (GLP-1R) is an iridoid glycoside from the fruit of Gardenia jasminoides Ellis used as a Chinese traditional medicine for treatment of vitiligo vulgaris. Interaction of c-kit receptor with its ligand-SCF potent enhances the melanocytic melanogenesis, which can be repressed by norepinephrine (NE). To discover economic and efficient drug against vitiligo vulgaris, this paper addresses the action and mechanism of GP abrogating the NE-induced hypopigmentation in melanocyte. Flow cytometry exhibited the up-regulation effect of GP on NE-suppressed production of c-kit by normal human epidermal melanocyte (HEMn) in a concentration-dependent manner, and exendin-(9-39) (selective GLP-1R antagonist) appeared to alleviate the GP-stimulated expression of c-kit. However, neither NE nor GP affected the production of SCF by normal human epidermal keratinocyte (HEKn) assessed by cellular enzyme-linked immunosorbent assay. Spectrophotometry documented that GP abrogated the repression effect of NE on tyrosinase activity and melanin production in HEMn in the presence of recombination SCF significantly. The response of melanocytic melanogenesis to GP was blocked by exendin-(9-39) or K44.2 antibody (c-kit inhibitory antibody). Data from this paper provide the evidence that GP abrogates the NE-induced hypopigmentation by the activation of GLP-1R-dependent c-kit receptor signaling in which c-kit expression is augmented in HEMn. [Copyright &y& Elsevier]

Objective In order to investigate the pharmacological properties of Ginkgo biloba extract (GBE) on improving blood circulation, the regulating action of GBE and quercetin (a main flavonoid ingredient in GBE) on thrombomodulin (TM) expression and tissue-type plasminogen activator (t-PA) secretion was studied. Methods Using flow cytometer and gel image system respectively, we evaluated the TM expression and the t-PA secretion by human umbilical vein endothelial cells (HUVECs) in vitro. Results The increase of TM expression on HUVECs surface was induced by GBE rather than quercetin in a dose- and time-dependent manner. Both GBE and quercetin increased the t-PA release significantly. Conclusion The effect of GBE on improving blood circulation may be partly attributed to its promoting TM expression and t-PA secretion by endothelial cells, and quercetin participated in the effect of OBE on t-PA secretion. However, the action of GBE on increasing TM expression needs further study. [ABSTRACT FROM AUTHOR]