[Study on antiangiogenesis effect of Vaccaria segetalis].

OBJECTIVE: To study the inhibiting effect of Vaccaria segetalis extracts on neovascularization. METHODS: The effect of Vaccaria segetalis extracts on the proliferation, migration in vitro and tube formation on Matrigel of endothelial cell (HMEC) in vivo were examined by MTT assay and Matrigel plug assay. RESULTS: The proliferation and migration of HMEC were inhibited significantly by Vaccaria segetalis extracts in a dose-dependent manner (IC50 = 50 microg/mL). It also inhibited angiogenesis in Matrigel plug mouse model. CONCLUSION: Vaccaria segetalis extracts can inhibit angiogenensis obviously, and it could be developed as an effective antiangiogenic drug.

Identification of binding peptides of the ADAM15 disintegrin domain using phage display.

ADAM15 plays an important role in tumour development by interacting with integrins. In this study, we investigated the target peptides of the ADAM15 disintegrin domain. First, we successfully produced the recombinant human ADAM15 disintegrin domain (RADD) that could inhibit melanoma cell adhesion by using Escherichia coli. Second, four specific binding peptides (peptides A, B, C, and D) were selected using a phage display 12-mer peptide library. The screening protocol involved 4 rounds of positive panning on RADD and 2 rounds of subtractive selection with streptavidin. By using the BLAST software and a relevant protein database, integrin alpha v beta 3 was found to be homologous to peptide A. Synthetic peptide A had a highly inhibitory effect on RADD-integrin alpha v beta 3 binding. The results demonstrate the potential application of short peptides for disrupting high-affinity ADAM-integrin interactions.

Anti-angiogenic activity of julibroside J8, a natural product isolated from Albizia julibrissin.

PURPOSE: The purpose of this study was to investigate the anti-angiogenic properties of julibroside J(8), a triterpenoid saponin isolated from Albizia julibrissin. METHODS: In the presence of juliborside J(8,) the growth of human microvascular endothelial cells (HMEC-1), four human tumor cell lines, and a normal cell line (MRC-5) was evaluated by MTT assay. The in vivo anti-angiogenic effect of julibroside J(8) was evaluated on a chorioallantoic membrane (CAM) and in transplanted colon carcinoma cells in a nude mice neovascularisation model. RESULTS: Treatment with 0.5-4 microg/ml julibroside J(8) resulted in dose-dependent inhibition of growth, migration, and tube formation in HMEC-1 cells; julibroside J(8) also inhibited the formation of microvessels on CAM at a concentration of 10-50 microg/egg and reduced vessel density within tumor at a concentration of 0.5-3mg/kg. CONCLUSIONS: Julibroside J(8) may be a potent anti-angiogenetic and cytotoxic drug; further investigation is warranted.

Expression, purification, and characterization of recombinant human serum albumin fusion protein with two human glucagon-like peptide-1 mutants in Pichia pastoris.

Glucagon-like peptide-1 (GLP-1) is a 30-residue peptide hormone secreted by intestinal L-cells in response to nutrient ingestion. In the present study, overlapping PCR technology was employed to construct two GLP-1 mutants (GLP-1(A2G))2 and human albumin (HSA) genes in vitro without linker. The spliced gene, (GLP-1(A2G))2-HSA, was over expressed under the control of promoter AOX1 and Mat alpha signal peptide in Pichia pastoris. SDS-PAGE and Western blotting were applied to assay the recombinant fusion protein in the culture broth. The results demonstrated that the recombinant (GLP-1(A2G))2-HSA concentration in the broth could reach a level of 245.0 mg/L and the expressed fusion protein was capable of cross-reacting with anti-human GLP-1 and anti-human albumin antibody. The recombinant (GLP-1(A2G))2-HSA protein was purified by ultrafiltration, columns of Q-sepharose fast flow and Superdex 75 size-exclusion. The recombinant (GLP-1(A2G))2-HSA protein obtained could lower in vivo glucose concentration in blood and stimulate in vitro islet cell proliferation. In mouse model, the fusion protein was detectable in plasma even 308 h after a single subcutaneous dose of 1.25 mg/kg. The result showed that the terminal biological half-time of the protein was about 54.2 h which is 650-fold longer than that of GLP-1. The pharmacokinetic analysis of the protein suggests its promising application in clinical medicine.

A one-step automated high-radiochemical-yield synthesis of 18F-FECNT from mesylate precursor.

2Beta-carbomethoxy-3beta-(4-chlorophenyl)-8-(2-[(18)F]fluoroethyl)nortropane ((18)F-FECNT) is a potential dopamine transporter imaging agent. In this article, a new mesylate precursor was prepared and a one-step automated synthesis of (18)F-FECNT was developed. The mesylate precursor (4) was synthesized from 2beta-carbomethoxy-3beta-(4-chlorophenyl)tropane (1) by N-demethylation, hydroxyethylation followed by mesylation at a total yield of 47%. [(18)F]fluorination was performed by heating 4mg mesylate precursor and K[(18)F] in 1 ml acetonitrile at 90 degrees C for 20 min. The crude (18)F-FECNT was obtained with a radiolabeling yield of 48%. After purification by preparative high performance liquid chromatography (HPLC), the final (18)F-FECNT product was obtained with a radiochemical purity of 98.4% and a decay corrected radiochemical yield of 33+/-9% (and the uncorrected radiochemical yield was 19+/-5%, n=5). The duration of the total procedure was 80-90 min.

[Effect and mechanism of recombinant hirudin on fibrinolysis].

AIM: To study the effect of recombinant hirudin (rH) on tPA-induced fibrinolysis and the possible mechanism of its action. METHODS: The effect of rH on thrombin-fibrin complex (Th-Fn) was detected by 99mTc labeled rH. In the in vitro clot lysis, tPA as plasminogen activator, and recalcified plasma as plasminogen resource were used to study the influence of rH on fibrinolysis by detecting TAFIa, D-Dimer and FXIII. RESULTS: In a canine model of femoral artery thrombosis, a clear radioactivity strip was imaged in 30 - 60 min on a part image, and the femoral vein thrombosis developed at 30 min. rH efficiently inhibited clot regeneration. Addition of TM could inhibit clot lysis obviously, and CPI could shorten the delay of clot lysis which due to TAFIa. There was a dose-dependent relationship with TM concentration and TAFI activation. FXIII activation was inhibited by low concentration of rH ( < or = 0.2 u x mL(-1)), and the level of fibrinolysis product, D-Dimer, increased. CONCLUSION: rH could inhibit the thrombin binding to fibrin. rH inhibited the activation of TAFI and FXIII by combining with thrombin which resulted in enhancement of thrombolysis.

[Studies on active substance of anticancer effect in Polygonum cuspidatum].

OBJECTIVE: To supply the scientific basis of research and development of the medicinal value of Polygonum cuspidatum. METHODS: One composition was isolated from the roots of Polygonum cuspidatum by cytotoxicity based fractionation and identified by HPLC-MS, UV scanning and IR. The inhibition and morphology of L-02, Hep G2, SHZ-888, MCF-7, MCF-7/ADM cells growth caused by this composition was determined by MTT assay and HE dyeing. RESULTS: This composition was identified as trans-and cis-resveratrol. It could specifically inhibit proliferation of many cancer cells but not human normal liver cell. We investigated the cytotoxicity of resveratrol to adriamycin-resistant MCF-7 cell in virtro. CONCLUSION: Resveratrol is a new anticancer composition which is less toxicity and higher efficiency in Polygonum cuspidatum.

[Use of ultrasensitive time-resolved fluoroimmunoassay for rapid detection of clenbuterol hydrochloride in urine].

OBJECTIVE: In order to provide a rapid and selectivity method for the determination of clenbuterol(CBL), an indirect competitive time-resolved fluoroimmunoassay (TRFIA) was developed. METHODS: Anti-CBL antibody, was raised by immunization against CBL-BSA in rabbits. CBL-OVA was coated by physical adsorption onto the microtitre plate, CBL or sample with CBL as a competitor. Both them were incubated with limited anti- CBL antibody. and a goat antirabbit IgG-Eu3+ conjugate was used as a tracer. RESULTS: The sensitivity of CBL-TRFIA was 0.01microg/L, and the recovery rate was 99.7%. RSD of CBL-TRFIA was 3.9% . The sensitivity of CBL-TRFIA provided a linear response from 0.01 - 25microg/L, with ED50 of (1.47+/-0.11) microg/L or ED80 of (0.07+/-0.01)microg/L and ED, of (23.6+/- 0.56) microg/L. The cross reactivity of the CBL-TRFIA with salbutamol, epinephrine hydrochloride and epinephrine bitartrate was negligible, while that with isoprenaline hydrochloride was 0.01% . Both CBL-TRFIA and CBL-ELISA test were applied for the quantitative measurement of CBL in the same urine, and the coefficient of correlation was 0.932. CONCLUSION: The CBL-TRFIA could be applied to detect the CBL in urine and it is useful to screening easily for CBL contamination in meat or foods.