Dual pH and Temperature-Sensitive Nanogels Loaded with Eugenol for Regulating Central Nervous System.

Fragrances have many biological activities such as anti-anxiety, anti-depression, and improving cognitive memory. However, most fragrances are so volatile that the useful lifespan of the fragrances is very short and excessive fragrance concentration makes us uncomfortable. In this study, dual pH and temperature-sensitive nanogels named EG@CPMONGs were prepared to encapsulate eugenol. This nano-fragrance was then applied to silk. In the following, the effects of EG@CPMO-NGs on the regulation of central nervous systems were evaluated. Open-field tests showed that EG@CPMONGs had an obvious effect on stress relief. Elevated plus-maze tests proved the significant effect of EG@CPMO-NGs on anti-anxiety. Morris water maze tests demonstrated the positive impact of nano-fragrance on spatial learning and memory. Therefore, these dual pH and temperature-sensitive nanogels loaded with eugenol had significant and positive effects on the central nervous system.

[Secretory expression vector V-pLNCX-s-hri inhibits the growth of mouse B16 melanoma].

BACKGROUND AND OBJECTIVE: Human ribonuclease inhibitor (hRI) extracted and purified from human placenta has been shown to remarkably inhibit some solid tumors in mice. This study was to construct V-pLNCX-s-hri, a secretory expression vector, and explore its inhibition effects on the growth of mouse B16 melanoma cells. METHODS: The hRI gene sequence conjugated with the synthesized signal peptide of mouse IgG was cloned into the retroviral vector V-pLNCX to construct V-pLNCX-s-hri. The PA317 cells were used for viral package and NIH3T3 cells were employed to determine the viral titer. The expression of hRI gene was detected by RT-PCR and Western blot. The content of RI was determined by enzyme-linked immunoabsorption assay (ELISA). The model of B16 melanoma-carrying mouse was established and received different treatments. The tumor weight and microvessle density (MVD) were assessed. Normal saline (NS), V-pLNCX, and V-pLNCX-hri were used as controls. RESULTS: The infection efficiency of V-pLNCX-s-hri on cultured B16 cells reached 38.5%. mRNA and protein levels of hRI were detected in B16 cells infected by V-pLNCX-s-hri. The hRI content in the supernatant of infected B16 cells reached 0.228 microg/mL. The hRI content in the peripheral blood of experimental mice was significantly higher in the V-pLNCX-s-hri group (0.249 microg/mL) than in the NS group (0.035 microg/mL), V-pLNCX group (0.028 microg/mL) and V-pLNCX-hri group (0.169 microg/mL) (P<0.01). The tumor weight and MVD were significantly lower in the V-pLNCX-s-hri group compared with those in the NS, V-pLNCX and V-pLNCX-hri groups (P>0.01). CONCLUSIONS: V-pLNCX-s-hri can effectively infect B16 cells and induce high expression of hRI. V-pLNCX-s-hri is superior to V-pLNCX-hri in inhibiting the growth of B16 cells.

[Anti-oxidative effect of ribonuclease inhibitor by site-directed mutagenesis and expression in Pichia pastoris].

Human placental ribonuclease inhibitor is an acidic protein of Mr approximately 50 kDa with unusually high contents of leucine and cysteine. It is a cytosolic protein that protects cells from the adventitious invasion of pancreatic-type ribonuclease. HRI has 32 cysteine residues, and the oxidative formation of disulfide bonds from those cysteine residues is a rapid cooperative process that inactivates HRI. The most proximal cysteine residues in native HRI are two pairs that are adjacent in sequence. In the present paper, two molecules of alanine to substitute for cys328/cys329 were performed by site-directed mutagenesis. The site-mutated RI cDNA was constructed into plasmid pPIC9K, and then transformed Pichia pastoris GS115 by electroporation. After colony screening , the bacterium was cultured and the product was purified with affinity chromatography. The affinity of the recombinant human RI with double site mutation was examined for RNase A and its anti-oxidative effect. The results indicated that there was no much change in the affinity for RNase A detected when compared with the wild type of RI. But the capacity of anti-oxidative effect was increased by 7-9 times. The enhance in anti-oxidative effect might be the reason for preventing the formation of disulfide bond between cys328 and cys329 and the three dimensional structure of RI was thereby maintained.

Cloning and characterization of Adinbitor, a novel disintegrin from the snake venom of Agkistrodon halys brevicaudus stejneger.

Adinbitor was cloned from Agkistrodon halys brevicaudus stejneger and characterized as a novel disintegrin. In this study, total RNA was extracted from venom gland and used in RT-PCR to generate a cDNA which is 219 bp long. The sequence encoded a polypeptide composed of 73 amino acids, including 12 cysteines, an RGD motif, and the signature motif of disintergrin. Recombinant Adinbitor (rAdinbitor) was expressed in E. coli and purified by using the His. Bind affinity chromatography. The IC(50) for inhibiting human platelet aggregation and bFGF-induced proliferation of ECV304 cells was 6 microM and 0.89 microM respectively. Furthermore, Adinbitor significantly inhibited angiogenesis both in vivo and in vitro. Taken together, these results suggested that Adinbitor had typical functions of disintegrins.

[New approach to resolution enhancement of Fourier transform spectrum].

Treating resolution enhancement of Fourier transform spectrum as the estimations of harmonic frequencies and amplitudes in the interferogram, the method based on eigenvalue analysis and linear fitting (EALF) was brought forward. EALF was divided into three steps: parameter estimation with total least squares, frequency estimation with eigenvalue analysis, and amplitude estimation with least squares fitting. The principle of the method was introduced. Computer experiment was presented to validate the principle. Characteristics of the method were discussed. The main advantages of EALF over alternative approaches to resolution enhancement of Fourier transform spectrum were summarized.

The antioxidant effects of ribonuclease inhibitor.

Ribonuclease inhibitor (RI) is an acidic cytosolic glycoprotein with molecular weight of about 50 kDa, which contains 32 cysteine residues. It is possibly that RI may have antioxidant effect by thiol-disulfide exchange reaction. We studied the effects of RI over-expression on the rat glial cell line C6 injured with H2O2. The transfected C6 cells with RI cDNA (C6') had higher viability, less LDH leakage and MDA contents, but more GSH contents compare that in the control C6 cells. In transfected C6 cells, the activities of CAT and GST were higher than that in the control C6 cells. Without H202 stress, the activities of CAT and GST in the C6' cells were 1.73 and 3.62 times that in the control C6 cells, respectively; With 1.00 mmol/L H2O2 stress, the activities of CATand GSTin the C6' cells were 3.38 and 2.11 times that in the C6 cells, respectively. These results suggest that the over-expression RI has antioxidant activity and it is able to protect cells from per-oxidative injuries. Moreover, we investigated whether RI has a protective role against mouse hepatic damage in vivo. The mice pretreated with different doses of human RI were injected by CC14. The results show that the SOD activities of therapy groups were significantly higher than that of the control group (p < 0.01), while the contents of MOD and activities of ALT and AST in blood were remarkably lower than that of the control group (p < 0.01). Pathological examination shows that the degree of damage was alleviated with RI therapy. These results suggest that RI has the protective role against mouse hepatic damage induced by CC14. The anti-oxidative effects of RI may play an important role in cell protection from per-oxidative injuries.