HIV-1 gains entry into target cells by sequentially interacting with cellular receptors and co-receptors. Both the receptor and co-receptor are recognized by HIV-1 envelope protein gpl20, which plays a key role in the entry process of HIV-1 into cells. The development of new inhibitors is essential since the viral enzyme reverse transcriptase （RT） is one of the first targets of antiretroviral therapy. It has been reported that a variety of natural plants, such as Artemisia rupestris L., have anti-viral pharmacological activity, and they might be the potential inhibitors of RT or V3 loop of gpl20 against HIV-1. RIQRGPGRAFVT1GK （R15K）, the relatively conserved region of V3 loop, can be used for binding research. In this work, we analyzed the interactions between different extracts from Artemisia rupestris L. and R15K by affinity capillary electrophoresis （ACE）. Moreover, we analyzed the interactions between different extracts from Artemisia rupestris L. and RT by capillary zone electrophoresis （CZE）. Our data showed that the chloroform extract ofArtemisia rupestris L. was active among the different plant extracts, which was consistent with previous studies. Taken together, our study provided a rapid screening method to seek anti-HIV ingredients in natural plants＇ extracts.
V3 loop of HIV-1 envelop protein gp120 plays a pivotal role in the entry process of HIV-1 into target cells. R15K, the relatively conserved region of V3 loop, can be used in binding studies instead of recombinant gp120 molecule. Polyanionic compounds, such as carrageenan, possess antiviral activity through disrupting gp120-CD4 interaction, and chemical modifications have been performed to improve such activity. In this work, we, for the first time, analyzed the interactions between carrageenan or its degradation and R15K by affinity capillary electrophoresis （ACE）. Our results revealed that depolymerized carrageenan rather than carrageenan could bind to R15K. The binding constant of depolymerized carrageenan was （2.94±0.57）× 10^6 mol/L. Our finding indicated that the depolymerized carrageenan could be R15K antagonist, and it might inhibit the infection of HIV-1 through the entry process.
A sensitive and specific high performance liquid chromatography method was established for measuring cefazolin sodium level in rabbit synovial fluid. Acetonitrile was used to precipitate proteins and antipyrine was used as internal standard. Samples were analyzed on a Dionex Ultimate U3000 HPLC system equipped with Phenomenex Luna C18 column （150 mmx4.60 mm, 5 Bm, 100 A）. The mobile phase consisted of acetonitrile and 0.1% formic acid in water. The flow rate was 1 mL/min. The detection wavelength was set at 272 nm. The column temperature was maintained at 25 ℃. The calibration curve was linear in the range of 1.0-100.0 μg/mL （r2 = 0.9999）. The limit of detection （LOD, S/N - 3） was 0.07 μg/mL. The limit of quantitation （LOD, S/N = 10） was 0.22 pg/mL. The recovery of cefazolin sodium （low, medium and high） was 124.6%, 117.8%, and 100.6% （RSD% - 1.9%, 4.0%, 1.1%, n = 5）, respectively. The intra-day and inter-day precision values were in the range of 0.5%-2.7%. The method was simple, sensitive and reliable. It can be used for the quantitative determination of cefazolin sodium in rabbit synovial fluid.
CC chemokine receptor 4（CCR4） is a G-protein-coupled receptor which plays a pivotal role in allergic inflammation. In the present study, three extracellular loops（EL1-3） of CCR4 were synthesized, and the interactions between the extracellular loops and compound S009 were investigated using capillary zone electrophoresis（CZE）. Both qualitative and quantitative characterizations of the compound-peptide binding were carried out. The experimental data indicated that compound S009 exhibited interactions with EL3, and a binding constant of（12.5±0.19）×10^4 M^-1 was determined using the Scatchard plot. Our study identified the specific domains of CCR4 that could be targeted by small molecules and provided insights for the discovery of novel CCR4 antagonists.
Abstract： In the presem study, we simultaneously quantified the levels of monoamine neurotransmitters （MANTs） and their metabolites （levodopa, norepinephrine, epinephrine, dopamine, 5-HT, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindole-3-acetic acid） in different brain subregions of rats using a newly developed simple, sensitive and selective high-performance liquid chromatography with fluorescence detection （HPLC-FLD） method. In this new HPLC-FLD method, analytes were directly extracted and separated without deriveatization step within 20 min. The FLD wavelength was set at 280 nm and 330 nm for excitation and emission, respectively. The analytes were separated on an Agilent Eclipse Plus Cls column （4.6 mm×150 mm, 5.0 μm） equipped with an Agilent XDB-C18 security guard column （4.6 mm×12.5 mm, 5.0 lam）, and the column temperature was maintained at 35 ℃. The mobile phase for elution was isocratic. The mobile phase consisted of citric acid buffer （50 mmol/L citric acid, 50 mmol/L sodium acetate, 0.5 mmol/L octane sulfonic acid sodium salt, 0.5 mmol/L Na2EDTA and 5 mmol/L triethylamine, pH 3.8） and methanol （90：10, v/v） at a flow rate of 1.0 mL/min. The detection limit （DL） was 0.9-23 nM for all the MANTs and their metabolites with a sample volume of 50 μL. The method was shown to be highly reproducible in terms of peak area （intraday, 0.08%-1.85% RSD, n = 5）. The simultaneous measurement of these MANTs and their metabolites improved our understanding of the neurochemistry in the central nervous system （CNS） in relation to different addictive drugs （methamphetamine, heroin and their mixture） in drug-addicted rat models.
A rapid and simple liquid chromatography method with on-line solid phase extraction was developed and validated for the quantitative determination of cyclophosphamide in rat plasma.The plasma sample was first extracted on an Acclaim？ Polar Advantage II C18 guard column（PA II C18,10 mm×4.6 mm,5 μm）,which was also the on-line Extraction Cartridge SPE column,by washing with 100% H2O for 1 min.The extracted sample was then eluted onto a PA II C18 column（150 mm×4.6 mm,5 μm） and separated by isocratic elution with acetonitrile-water（40：60,v/v）.The mobile phase was run at a flow rate of 1.0 mL/min,and the UV detector was set at 195 nm.Retention time of cyclophosphamide was 4.3 min and the total run-time was 6 min.The linear range of the standard curve was from 1.0 to 200 μg/mL（r2 = 0.9999）,and the limits of quantification and detection were 1.0 μg/mL（RSD10%,n = 5） and 0.3 μg/mL（RSD13%,n = 5）,respectively.Both intra-and inter-day variations were less than 5.6%.The developed method can be used for the therapeutic drug monitoring of cyclophosphamide in the clinic.
