Improved protein purification system based on C-terminal cleavage of Npu DnaE split intein.

A purification system was constructed with the N-segment of the Npu DnaE split intein as an affinity ligand immobilized onto an epoxy-activated medium and the C-segment used as the cleavable tag fusing target protein. The affinity properties of C-tagged proteins adsorbed on IN affinity chromatography medium were studied with GFP as a model target protein. The saturated adsorption capacity and dynamic adsorption capacity reached 51.9-21.0 mg mL-1, respectively. With this system, two model proteins, GFP and alcohol dehydrogenase (ADH), has been successfully taglessly purified with regulation of Zn2+ and DTT. The yield, purification factor and purity of purified tagless GFP reached 39, 11.7 and 97%, respectively; while these values for purified tagless ADH were 38.2, 6.8 and 91%, respectively. These results showed that the system for Npu DnaE split intein-mediated affinity adsorption and in situ cleavage is a potential platform for recombinant protein production.

The correlation between the length of repetitive domain and mechanical properties of the recombinant flagelliform spidroin.

Spider silk is an attractive biopolymer with numerous potential applications due to its remarkable characteristics. Among the six categories of spider silks, flagelliform (Flag) spider silk possesses longer and more repetitive core domains than others, therefore performing the highest extensibility. To investigate the correlation between the recombinant spidroin size and the synthetic fiber properties, four recombinant proteins with different sizes [N-Scn-C (n=1-4)] were constructed and expressed using IMPACT system. Subsequently, different recombinant spidroins were spun into fibers through wet-spinning via a custom-made continuous post-drawing device. Mechanical tests of the synthetic fibers with four parameters (maximum stress, maximum extension, Young's modulus and toughness) demonstrated that the extensibility of the fibers showed a positive correlation with spidroin size, consequently resulting in the extensibility of N-Sc4-C fiber ranked the highest (58.76%) among four fibers. Raman data revealed the relationship between secondary structure content and mechanical properties. The data here provide a deeper insight into the relationship between the function and structure of Flag silk for future design of artificial fibers.

Phloroglucinol protects the urinary bladder via inhibition of oxidative stress and inflammation in a rat model of cyclophosphamide-induced interstitial cystitis.

BACKGROUND: Phloroglucinol plays an important role in oxidative stress and inflammatory responses. The effects of phloroglucinol have been proven in various disease models. The aim of the present study was to investigate the efficacy and possible mechanisms of phloroglucinol in the treatment of interstitial cystitis (IC). METHODS: Thirty-two female Sprague-Dawley (SD) rats were used in this study. IC was induced by intraperitoneal injection of cyclophosphamide (CYP). Rats were randomly allocated to one of four groups (n = 8 per group): A control group, which was injected with saline (75 mg/kg; i.p.) instead of CYP on days 1, 4, and 7; a chronic IC group, which was injected with CYP (75 mg/kg; i.p.) on days 1, 4, and 7; a high-dose (30 mg/kg) phloroglucinol-treated group; and a low-dose (15 mg/kg) phloroglucinol-treated group. On day 8, the rats in each group underwent cystometrography (CMG), and the bladders were examined for evidence of oxidative stress and inflammation. Statistical analysis was performed by analysis of variance (ANOVA) followed by least square difference multiple comparison post-hoc test. RESULTS: Histological evaluation showed that bladder inflammation in CYP-treated rats was suppressed by phloroglucinol. CMG revealed that the CYP treatment induced overactive bladder in rats that was reversed by phloroglucinol. Up-regulated tumor necrosis factor-α and interleukin-6 expression in the CYP-treated rats were also suppressed in the phloroglucinol treated rats. CYP treatment significantly increased myeloperoxidase activity as well as the decreased activities of catalase of the bladder, which was reversed by treatment with phloroglucinol. CONCLUSIONS: The application of phloroglucinol suppressed oxidative stress, inflammation, and overactivity in the bladder. This may provide a new treatment strategy for IC.

[Effects of 2000 µW/cm2; electromagnetic radiation on expression of immunoreactive protein and mRNA of NMDA receptor 2A subunit in rats hippocampus].

AIM: To evaluate the effects of electromagnetic irradiation of 2000 µW/cm(2); exposure on mRNA and protein expression levels of immunoreactive protein and mRNA of NMDA receptor 2A subunit in rats hippocampal, and to explore the mechanism of electromagnetic irradiation induced learning and memory impairment. METHODS: Rats were randomly divided into normal control group, sham-radiated group, and 1 h/d, 2 h/d, and 3 h/d radiation groups. The rats in the radiation groups were fixed after microwave exposure of 2000 µW/cm(2);, then their learning and memory abilities were tested by Morris water maze experiment, the change of NR2A protein in hippocampal neurons of each group of rats were measured with immunohistochmistry and Western blot techniques, and the expression of NR2A mRNA in hippocampus were determined by RT-PCR. RESULTS: Compared with the normal control group, each index of the sham-radiated group has no significant change (P>0.05), while the latency of rats of radiated group in Morris water maze test were significantly longer (P<0.05). In the radiation group, the hippocampal neurons of rats showing evident reduction in the ratio of NR2A positive cells, irregular, and arrayed in disorder. Moreover, the expession of NR2A protein and its mRNA in hippocampal neurons were significant decreased (P<0.05). CONCLUSION: Electromagnetic irradiation of 2000 µW/cm(2); exposure can impair the learning and memory abilities of rats possibly through a mechanism correlated with the lower expression of NR2A protein and its mRNA in hippocampus.

Selective determination of trace 17ß-estradiol in dairy and meat samples by molecularly imprinted solid-phase extraction and HPLC.

Molecularly imprinted solid-phase extraction (MISPE) combined with HPLC were used to detect trace 17ß-estradiol (E2) in different dairy and meat samples. E2 imprinted mono-dispersed microspheres prepared by modified precipitation polymerisation method were used as MISPE sorbents. Under optimum MISPE and HPLC conditions, trace (0.116-0.461nmolkg(-1)) E2 in different dairy and meat samples can be detected accurately using UV detector when large amount of sample (500g) was analysed. Recoveries of E2 from spiked (0.1-5nmolkg(-1)) milk, yogurt, beef, pork and chicken were higher than 74.18%, 71.92%, 67.21%, 64.20% and 65.93%, respectively with RSDs less than 8.38%. MISPE can enrich E2 selectively, which is helpful to have better HPLC separation and higher recoveries than C18 SPE. MISPE combined with HPLC-UV are reliable methods to determine trace E2 in dairy and meat samples with high accuracy and repeatability.

Selective molecularly imprinted stationary phases for bisphenol A analysis prepared by modified precipitation polymerization.

Bisphenol A (BPA)-imprinted polymeric microspheres were synthesized by modified precipitation polymerization (MPP) method. Influences of cross-linker, monomer, porogen volume, and agitation on polymerization were investigated. Proper amount of cross-linker ethyleneglycol-dimethacrylate (EGDMA) was critical to achieve narrowly dispersed microspheres. For template BPA, monomer 4-vinylpyridine (4-VP) was better than MAA to get the best imprinted effects. The optimum template/monomer ratio was 1:6. Increasing porogen volume increased size dispersity and decreased binding characters. Agitation increased coagulation and resulted in irregular particles. Microspheres with the best binding characters were used as selective stationary phase of chromatographic column to detect BPA in milk, pig urine, and chicken meat. Under optimal chromatographic conditions, the calibration graph was linear with R2 = 0.9994 in the range of 3-50 micromol/L. The LOD and LOQ were 1 and 3 micromol/L, respectively. When large amounts (20 mL or 20 g) of samples were analyzed, the recoveries ranged from 70.2 to 87.3% with RSD less than 4.85% in all samples spiked with 0.05-0.2 micromol/L BPA. The intra-day and inter-day RSD were less than 1.83 and 3.96%, respectively. Microspheres prepared by MPP are successfully used in molecularly imprinted polymer (MIP)-based analytical column to detect trace BPA in different biologic samples with acceptable accuracy and repeatability.

[Revaluation of soil heavy metals pollution in Zhangshi irrigation area of Shenyang and analysis of Cd forms in soil].

This paper revaluated the situation of cadmium (Cd) pollution in rice field soils of Zhangshi irrigation area in Shenyang. The results indicated that the soil Cd pollution of test area was still quite serious. The Cd content in brown rice samples ranged from 0.435 to 0.855 mg x kg(-1), which exceeded the national criteria for food sanitation in China, and increased by 335%-755% compared with that 20 years ago. Brown rice Cd had a significant negative correlation with soil pH, and a positive correlation with soil available Cd. The decrease of soil pH induced more soil Cd transformed into available Cd which occupied 22.8%-52.0% of the total Cd and was easy to be absorbed by rice plant, inducing the Cd in brown rice exceeded national criteria. The pollutant Cd could transfer along with surface runoff, ground water and flying dust, making the pollution extend gradually.