人肝癌SMMC-7721细胞对低剂量γ射线辐射超敏感性的研究

研究了人肝癌细胞SMMC-7721在低剂量γ射线照射下超敏感性和增强的辐射抗性响应。选用对数生长期细胞接受0—6 Gy不同剂量的^60Coγ射线的照射。利用流式细胞仪对细胞进行分选计数,并用克隆形成法检测细胞存活率。发现SMMC-7721细胞存在低剂量辐射超敏感性和增强的辐射抗性响应,即在0—0．3Gy之间细胞表现出单位剂量杀伤增强现象,在0．3—1Gy细胞表现一定的辐射抗性,在1Gy以上,细胞的存活符合线性平方模型。     

Inhibitory Effect of Lithospermic Acid on the HIV-1 Nucleocapsid Protein

The HIV-1 nucleocapsid protein (NC) is a desirable target in antiretroviral therapy due to its high conservation among HIV-1 strains, and to its multiple and crucial roles in the HIV-1 replication cycle. Natural products represent a valuable source of NC inhibitors, with the catechol group being a privileged scaffold in NC inhibition. By coupling molecular modeling with NMR spectroscopy and fluorescence-based assays, we disclosed lithospermic acid, a catechol derivative extracted from Salvia miltiorrhizza, as a potent and chemically stable non-covalent inhibitor of the NC. Being different from other catechol derivative reported so far, lithospermic acid does not undergo spontaneous oxidation in physiological conditions, thus becoming a profitable starting point for the development of efficient NC inhibitors.     

Chemical composition and antioxidant activity of aerial parts of Ferula longipes Coss. ex Bonnier and Maury

This is the first study on the phytochemistry and antioxidant activity of Ferula longipes Coss. ex Bonnier and Maury (Apiaceae). A new flavonoid quercetin-3-O-α-L-rhamnopyranoside-7-O-ß-D-[2-O-caffeoyl]-glucopyranoside (1), along with 10 known compounds kaempferol-3-O-α-L-rhamnopyranoside (2), quercetin-3-O-α-L-rhamnopyranoside (3), kaempferol-3-O-ß-D-glucopyranoside-7-O-α-L-rhamnopyranoside (4), isorhamnetin-3-O-α-L-rhamnopyranoside-7-O-ß-D-glucopyranoside (5), quercetin-3-O-α-L-rhamnopyranoside-7-O-ß-D-glucopyranoside (6), isorhamnetin-3,7-di-O-β-D-glucopyranoside (7), apigenin (8), apigenin-7-O-ß-D-glucopyranoside (9), 3,5-dicaffeoylquinic acid (10), deltoin (11) were isolated from the aerial parts of Ferula longipes Coss. Structures elucidation was performed by comprehensive 1D and 2D NMR analyses, mass spectrometry and by comparison with literature data. The compounds 1, 3, 4, 6, 7 and 10 were evaluated for their antioxidant activity, compound 1 exhibited the best antiradical activity potential and showed IC₅₀ and A_0.5 values 5.70, 7.25, 5.00, and 2.63 μg/mL towards DPPH free radical-scavenging, ABTS, CUPRAC, and reducing power assays, respectively compared with BHA, BHT and ascorbic acid which were used as positive controls.     

Odorant Receptor 7D4 Activation Dynamics

Deciphering how an odorant activates an odorant receptor (OR) and how changes in specific OR residues affect its responsiveness are central to understanding our sense of smell. A joint approach combining site‐directed mutagenesis and functional assays with computational modeling has been used to explore the signaling mechanics of OR7D4. In this OR, a genetic polymorphism affects our perception of androstenone. Molecular simulations totaling 0.12 ms predicted that, similarly to observations for other G‐protein‐coupled receptors with known experimental structures, an activation pathway connects the ligand and the G‐protein binding site. The 3D model activation mechanism correlates with in vitro data and notably predicts that the OR7D4 WM variant is not activated. Upon activation, an OR‐specific sequence motif is the convergence point of the mechanism. Our study suggests that robust homology modeling can serve as a powerful tool to capture OR dynamics related to smell perception.     

Performance of electrolyte assays in the United Kingdom—reference target values

Wales External Quality Assessment Scheme (WEQAS) is one of the largest external quality assessment (EQA) providers in the United Kingdom, with over 600 participants. Over the last two and a half years, reference target values have been used for the electrolyte panel in the WEQAS General Chemistry scheme. Reference methods have a vital role in that they ensure the transfer of accuracy from definitive methods to routine methods. Validated reference methods, using flame emission/atomic absorption spectrometry were established for sodium, potassium, calcium, magnesium and lithium. These methods were validated by direct comparison with the definitive method via the use of primary reference material. Comparison against the all laboratory trimmed mean (ALTM) showed a small mixed systematic error for sodium and potassium, with a maximum bias from the reference value of 1% and 2%, respectively. Lithium and magnesium data showed predominant systematic constant errors of +0.03 and +0.02 mmol/l, respectively. For calcium, ALTM results showed a mixed systematic error with a cross over in the reference range (5% positive bias and constant error of –0.1 mmol/l). Over 40% of participants used the cresolpthalein complexone method, greatly influencing the ALTM value, (11% bias, constant error –0.26 mmol/l). Other methods such as the ion selective electrode (ISE) method were in closer agreement to the reference method (–4% bias, +0.05 mmol/l constant error). The study highlights the pitfalls of using the overall mean data as an accuracy target in EQA schemes.Presented at the 8th Conference on Quality in the Spotlight, 17–18 March 2003, Antwerp, Belgium     

Enzyme-linked flow-injection immunoassay using immobilized secondary antibodies

A fast, non-equilibrium enzyme-linked flow-injection immunoassay (FIIA) system using an immobilized secondary-antibody reactor is described. The assay method is based on the competition between the enzyme-labeled antigen and analyte (unlabeled antigen) for a limited amount of soluble primary-antibody binding sites. This mixture is then introduced via flow-injection into the secondaryantibody reactor. The reactor bound enzyme activity, as measured by flowing an appropriate substrate solution through the reactor, is inversely proportional to the concentration of free analyte in the sample. By using non-equilibrium conditions, a single assay takes a total time of 13 min or less including regeneration of the reactor. To illustrate the application of this system, theophylline and insulin were chosen as model hapten and macromolecule analytes, respectively. Preliminary studies suggest that the new FIIA system is suitable for determining theophylline in serum with acceptable accuracy and precision.     

Comprehensive stability‐indicating high‐performance liquid chromatography coupled with diode array detection method for simultaneous determination of amprolium hydrochloride and ethopabate in powder dosage form for veterinary use

This research deals with the development of a stability‐indicating high‐performance liquid chromatography method for simultaneous determination of amprolium hydrochloride and ethopabate. To the best of our knowledge, no comprehensive stability‐indicating method has been reported for analysis of this mixture. Separation was achieved using Kromasil cyano column with gradient elution of the mobile phase composed of sodium hexane sulfonate solution and methanol. Quantification was based on measuring peak areas at 266 nm. Amprolium and ethopabate peaks eluted at retention times 10.42 and 18.53 min, respectively. The proposed procedure was validated with respect to system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection, and quantification limits. Linearity ranges for amprolium and ethopabate were 1.5–240 and 1–160 μg/mL, respectively. Analytes were subjected to stress conditions of hydrolysis, oxidation and thermal degradation. The proposed method enabled resolution of drugs from their forced‐degradation products and amprolium related substance (2‐picoline). Moreover, specificity was verified by resolution of the analytes from about 22 drugs used in antimicrobial veterinary products. The validated method was successfully applied to assay of the combined veterinary powder dosage form, additionally it was implemented in the accelerated stability study of the dosage form when stored for six months at 40°C and 75% relative humidity.     

Optimization of high expression and purification of recombinant streptokinase and in vitro evaluation of its thrombolytic activity

BackgroundStreptokinase (SK) is a potent plasminogen activator naturally produced by beta-hemolytic streptococcus bacteria and used as a thrombolytic drug.ObjectivesOptimize high yield production of recombinant streptokinase (rSK) in Escherichia coli and evaluate its thrombolytic activity.MethodsSynthetic gene encoding mature SK protein with optimization for rare codons and mRNA secondary structure was cloned into the expression vector pET-3a and transformed into Escherichia coli BL21 (DE3). Seed banks were established for high rSK expression clones. The native rSK protein expression was optimized using IPTG induction. The nonsoluble rSK inclusion bodies were purified, denatured in 6 M guanidinium chloride, and refolded using the rapid dilution method. The refolded rSK protein was purified using anion exchange chromatography and evaluated with ELISA. The activity of rSK was evaluated using the casein digestion method and in vitro blood clot lysis assay with reference drug Sedonase as standard.ResultsSeed banks with high stable expression of native rSk (MW 47 kDa) were established. High rSK expression was optimized using 1 mM IPTG at bacterial OD600 0.6. The refolded rSK was prepared and purified successfully with high productivity (494 mg purified rsk/L culture). Using ELISA, the purified rSK molecular identity and conservation of native SK epitopes were confirmed. The enzymatic activity of the purified rSK was 1.945x10⁶ IU/mg with 62.94 ± 2.3% clot lysis efficiency.ConclusionA high yield production of proper rSK protein with in vitro thrombolytic activity similar to commercial SK has been achieved, suggesting a more cost-effective industrial production of its biosimilar drug.     

Phytochemical profiling,molecular docking,and in vitro anti-hepatocellular carcinoid bioactivity of Suaeda vermiculata extracts

The ATP-binding cassette is the major class of transporters responsible for the efflux of chemotherapeutic agents from cancer cells, resulting in treatment failures of cancer’s patients. Suaeda vermiculata Forssk. ex. J. F. Gmel. is traditionally known for its liver protective activity. The LC-MS based chemical profilings of the sequentially partitioned sub-extracts obtained from the alcoholic extract of S. vermiculata using n-hexane, chloroform, ethyl acetate, and n-butanol as fractionating solvents, identified a total of thirty six compounds. These sub-extracts were evaluated for their anti-hepatocarcinoma activity against the sensitive HepG2 and doxorubicin (DOX)-resistant, HepG-2/ADR cell lines. A mixture of doxorubicin and sub-extracts at 20 μg/ml doses were also tested for their anti-hepatocarcinoma activity. The exhibited IC₅₀ values for the chloroform, ethyl acetate, n-hexane, and n-butanol sub-extracts, and the doxorubicin against HepG2, and HepG-2/ADR cell lines were found at 64.5, 66.8, 81.25, 125, 1.3 μg/ml, and 110.1, 91.82, 138.2, 265.7, 4.77 μg/ml levels, respectively. However, the treatment of resistant cells with 20 μg/ml of different sub-extracts in combination with the doxorubicin showed significant improvements in the doxorubicin activity against the resistant cells, and the IC₅₀ values for DOX + chloroform, DOX + ethyl acetate, DOX + n-hexane, and DOX + n-butanol against resistant cells, were at 1.77, 2.05, 2.66, and 2.71 μg/ml levels, respectively. The IC₅₀ values exhibited 2.69x, 2.33x, 1.79x and 1.76x-folds reversal of the sensitivity in the resistant cancer cell lines. The molecular docking studies of the compounds identified in the LC-MS chemical profilings, against three ATP-binding cassette proteins i.e., ABCB1, ABCC1, and ABCG2, showed that flavonoids as the major class of compounds responsible for reversal of the resistant cells sensitivities. The predicted binding affinity for the flavonoids against the above mentioned three ATP-binding cassette proteins’ are in the ranges of ～?8 to ?11 kcal/mol. Our results clearly indicate that the presence of flavonoids, as the major class of compounds in the S. vermiculata is responsible for the chemosensitization of the resistant HCC-cell lines. Moreover, the structures, 21 (5‐O‐methyl visamminol), 22 (N-trans-feruloyl tyramine), 27 (atractylenolide-III), and 32 (ginsenoside-Rh2) were also identified among the potential ATP-binding cassette’s modulators during the current study. These observations put the S. vermiculata in perspective with the traditionally claimed liver protective efficacy of the plant.