Construction of a recombinant eukaryotic expression vector containing DNM3 gene and its expression in colon cancer cells.

INTRODUCTION: Dynamin 3 (DNM3) is a large GTPase that possesses mechanochemical properties and has been shown to be involved in malignancies. However, most studies about DNM3 are observational, and knowledge of the precise molecular mechanism of DNM3 remains limited. MATERIALS AND METHODS: We constructed a PCDH-CMV-MCS-EF1a-GFP-Puro-DNM3 recombinant eukaryotic expression vector, which was then transfected into SW620 and LoVo cells. One cell line was divided into three groups. DNM3 mRNA and protein expression was analyzed by quantitative real-time PCR and Western blot assay. To investigate DNM3 biological activity in colon cancer SW620 and LoVo cell line, we performed cell proliferation, transwell migration, and invasion assay. Matrix metalloproteinase (MMP)-2 and MMP-9 protein expressions were detected by Western blot. RESULT: We successfully constructed a PCDH-CMV-MCS-EF1a-GFP-Puro-DNM3 recombinant eukaryotic expression vector, and stable DNM3 expression was observed in SW620 and LoVo cell lines. The vector overexpressing DNM3 inhibited the proliferation, weak invasion, and migration ability of colon cancer SW620 and LoVo cells relative to those in the control group (all P<0.001). DNM3 downregulated the protein expression of MMP-2 and MMP-9. CONCLUSION: DNM3 may weaken the malignant behavior of colon cancer and may have promoted the invasion and migration of colon cancer by regulating the expression of MMP-2 and MMP-9.

[Investigation of sulfonamides and tetracyclines antibiotics in soils from various vegetable fields].

The occurrence and distribution of 9 selected antibiotics, that involved with oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC), sulfamethazine (SM2), sulfadiazine (SDZ), sulfamerazine (SMR), sulfameter, (SMT), sulfamethoxazole (SMZ) and sulfadimethoxine (SDM), were screened at 14 typical vegetable fields located in Shenzhen, Guangzhou, Huizhou, Dongguan regions,with high-performance liquid chromatography (HPLC). Data showed that almost all soil samples were unavoidably contaminated with antibiotics. The sum concentration of 3 tetracyclines (TCs) and 6 sulfonamides (SAs) in soils ranged from undetected to 242.6 microg/kg and 33.3 to 321.4 microg/kg respectively, while the medium concentrations were 84.8 microg/kg of tetracyclines and 121 microg/kg of sulfonamides. The individual detectable probability of the two groups of antibiotics ranged from 19.35% to 96.77% (TCs) and from 25.81% to 93.50% (SAs). TC, SMZ, SMT and SM2 were more abundant in soil among the selected antibiotics. The study also suggested that the total amount of both TCs and SAs in different vegetable fields ranked as hoggery vegetable field > non-pollution vegetable field > routine vegetable field > greenfood vegetable field, and there should exist some relationship between vegetable planting and antibiotics amount in soil which was related to different fertilization, irrigation and vegetable species. The concentrations of the selected antibiotics are comparable to those reported from the other countries in the world, but the detectable probability of the antibiotics in our study site is higher than that from the reference data. More attention should be paid to antibiotics pollution of soil in vegetable fields.

Experimental spinal cord injury induced an increase of extracellular ascorbic acid concentration in anesthetized rats: a microdialysis study.

Ascorbic acid plays important roles in mammalian central nervous system. We employed an on-line analytical system to monitor the extracellular ascorbic acid concentrations in anesthetized rat spinal cord before and after the experimental injury. A microdialysis probe (216 microm od, 200 microm id, 3 mm in length) was implanted into an anesthetized rat spinal cord (Thoratic-12). Microdialysis perfusate (2 microl/min) was collected in the sample loop (20 microl) of an on-line injector for direct injection onto a High Performance Liquid Chromatography (HPLC) system equipped with an electrochemical detector. Normal ascorbic acid concentrations in the spinal cord extracellular fluids ranged from 1.8 microM to 10.8 microM (mean +/- S.D. 5.6 +/- 2.4 microM, n = 8). The experimental spinal cord injury, induced by a lesion at T-10, gradually yet significantly increased the extracellular ascorbic acid levels. The effect of exogenous glutamate perfusion (0.2 mM, 2 mM, and 20 mM) through the microdialysis probe also increased the extracellular ascorbic acid concentrations in a dose dependent manner. These results suggested that the injury-induced ascorbic acid accumulation may result from elevated extracellular glutamate levels that are commonly observed in spinal cord injury. This on-line, continuous and automatic monitoring system can be applied to future investigations on the roles of ascorbic acid in spinal cord injuries.