ABSTRACT
The analysis of stable nitrogen isotopic composition (δ15 N) of individual amino acid was recognized as an effective method for estimating the trophic level of organisms and detecting the nitrogen flow in food webs. In this study, we evaluated a two-stage procedure of esterification followed by acylation, in which biological samples underwent hydrolysis in acid and the released individual amino acids were derivative into the corresponding N-pivaloyl-iso-propyl ( NPP ) esters for gas chromatography-combustion-isotope ratio mass spectrometric ( GC-C-IRMS) analysis. A total of 13 kinds of individual amino acid derivatives were baseline separated on a nonpolar gas chromatography column (DB-5ms). The amount of sample for each test was not less than 20 ng N on column. High correlations were observed between the δ15 N values respectively obtained by GC-C-IRMS and element analysis-isotope ration mass spectrometry (EA-IRMS). Furthermore, the mean precision of this method was better than 1‰. Cation-exchange chromatograph was used to purify the samples, and the difference of the detection δ15 N values before and after purification by the resin was within 1‰. This method was applied to estimate the trophic level of various natural freshwater organisms from Aha Lake. The present study provided a new idea for the application of stable nitrogen isotope (δ15 N ) in the trophic level estimation of organisms and metabolism analysis of amino acid.
ABSTRACT
In the presence of silver ion, Mn2+ could be electro-oxidized to potassium hypermanganate in phosphoric acid solution, which could effectively react with pyrocatechol in acid solution and luminol in sodium hydroxide solution to produce chemiluminescence. On the basis of this, a novel indirect approach for the detection of Mn2+ was established. The effect of silver ions on the electrochemical oxidation of Mn2+was studied. when 1. 5 ×10-5 mol/L Ag+ and 0. 01 mol/L phosphoric acid solution were used in the process of electrochemical oxidation, the CL intensity could be up to the maximum value after the above solution was electrolyzed for 2 min. The relation of CL intensity and Mn2+concentration in the solutions at different pH and the selectivity were also investigated. when the pyrocatechol was used as luminescent reagent in the acidic medium, the CL intensity was linearly to the Mn2+concentration in the range of 1. 82×10-7-7. 27×10-5 mol/L with excellent selectivity. Common ions had little interferences in the determination of Mn2+. The method was successfully applied to the determination of Mn2+ in surface water and drinking water with satisfactory results.
ABSTRACT
AIM:To explore the effects of confluent endothelial progenitor cells (EPCs) derived from young and aged rats on the phenotype conversion, proliferation and migration of vascular smooth muscle cells ( SMCs) .METH-ODS:Mononuclear cells were obtained from the bone marrow of young (1~2 month old) and aged (19 to 26 month old) Sprague-Dawley rats and cultured with medium DMEM/F12 ( containing 15% fetal bovine serum, endothelial cell growth supplements (ECGs) 100 g/L, 1 ×105 units/L of penicillin and streptomycin, respectively).EPCs were characterized as double positive for DiI-Ac-LDL uptake and lectin binding.Abdominal aorta was obtained from 1 to 2 month old Sprague-Dawley rats.Vascular SMCs were cultured by tissue explant method and identified byα-SM-actin immunofluorescence.In transwell co-culture system, the confluent EPCs located in the upper chamber and SMCs were seeded on the lower cham-ber.The experiments were divided into passage 3 SMCs group (P3), passage 4 SMCs group (P4), passage 4 SMCs co-culture with EPCs derived from young rats group (P4YE) and passage 4 SMCs co-culture with EPCs derived from aged rats group (P4AE).The protein expression ofα-SM-actin and osteopontin was detected by Western blotting.[3H]-TdR incor-poration assay was used to determine the proliferation.SMC migration was analyzed by scratch wound healing assay.RE-SULTS:Compared with P3 group,α-SM-actin expression in P4 group significantly decreased and osteopontin protein ex-pression obviously increased, whereas no significant change was found in P4YE group.Compared with P4 group, confluent EPCs derived from young and aged rats both markedly increased α-SM-actin and decreased osteopontin expression in P4 SMCs.Compared with aged rat-derived EPCs, young rat-derived EPCs were more effectively to induce a delayed SMC phe-notype transition (from contractile phenotype to a synthetic phenotype), and to inhibit SMC proliferation and migration. CONCLUSION:Co-culture of confluent EPC induces a delayed vascular SMC phenotype transition and inhibits SMCs pro-liferation and migration.Young rat derived EPCs are more effective to induce a delayed vascular SMC phenotype transition and has stronger inhibitory effects on SMCs proliferation and migration compared with that derived from aged rats.
ABSTRACT
Objective To investigate the different influences of simvastatin on proliferation of rat smooth muscle progenitor cells(SPCs) versus endothelial progenitor cells (EPCs) and identify the compounds that differentially inhibit SPCs and EPCs proliferation for clinical usefulness. Methods Total mononuclear cells (MNCs) were isolated from bone marrow of rats by Fieoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. SPCs outgrew from the culture of MNCs in the presence of platelet-derived growth factor-BB and basic fibroblast growth factor, whereas EPCs were obtained in the presence of vascular endothelial growth factor. SPCs were identified as adherent cells positive for α-smooth muscle actin (α-SMA) by indirect immunofluoreseent staining. EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining. SPCs and EPCs were stimulated by simvastatin (0.01～10.00 μmol/L) or vehicle control for the respective time points (6 h, 12 h, 24 h and 48 h). SPCs and EPCs proliferation were assayed with 3H-TdR incorporation and manual counting respectively. Results Simvastatin obviously inhibited SPCs proliferation. At the concentration of 0. 01 μmol/L for 12 h,simvastatin significantly reduced the number of SPCs by (5.8±3.1)% compared with control group (P<0.05). Simvastatin significantly stimulated EPCs proliferation, which was dose- and time dependent and reached maximum at 1 μmol/L after 24 hours (2.0±0.1 fold increase, P<0.01).Conclusions Simvastatin displays different effects on SPCs (inhibited) and EPCs (promoted)proliferation. Local application of simvastatin may inhibit arterial restenosis and promote reendothelialization of injured vessels.