DNA loop sequence as the determinant for chiral supramolecular compound G-quadruplex selectivity.

It is important to develop G-quadruplex binding agents that can discriminate between different quadruplexes. Recently we reported the first example that a chiral supramolecular complex can selectively stabilize human telomeric G-quadruplex among different G-quadruplex and duplex DNA, and the two enantiomers show different inhibition effect on telomerase activity. Here, we report that DNA loop sequence can be determinant for this chiral complex G-quadruplex selectivity. Adenine in the diagonal loop plays an important role in G-quadruplex hybrid structural transition, thus, it strongly influences the chiral complex induced DNA structural transition. The complex's preference for human telomeric DNA and its chiral selectivity prompted us to investigate whether the two enantiomers, M and P, can show different effects on cancer cells. The P enantiomer's chiral selectivity has been demonstrated in cancer cells by telomere shortening, beta-galactosidase activity, and up-regulation of cyclin-dependent kinase inhibitors p16 and p21.

Chiral metallo-supramolecular complexes selectively recognize human telomeric G-quadruplex DNA.

Here, we report the first example that one enantiomer of a supramolecular cylinder can selectively stabilize human telomeric G-quadruplex DNA. The P-enantiomer of this cylinder has a strong preference for G-quadruplex over duplex DNA and, in the presence of sodium, can convert G-quadruplexes from an antiparallel to a hybrid structure. The compound's chiral selectivity and its ability to discriminate quadruplex DNA have been studied by DNA melting, circular dichroism, gel electrophoresis, fluorescence spectroscopy and S1 nuclease cleavage. The chiral supramolecular complex has both small molecular chemical features and the large size of a zinc-finger-like DNA-binding motif. The complex is also convenient to synthesize and separate enantiomers. These results provide new insights into the development of chiral anticancer agents for targeting G-quadruplex DNA.

Enhancement of immunity and resistance in mice by pig IL-6 gene and CpG motifs encapsulated in chitosan nanoparticle.

This study was conducted to explore the synergetic effect of a novel plasmid containing a porcine IL-6 gene and CpG motifs on immunity of mice in order to develop an effective adjuvant to boost resistance against infection. The synthetic oligodeoxynucleotide containing 11 CpG motifs was inserted into the reconstructed VR1020 plasmid containing the pig IL-6 gene (VRPIL6), designated VRIL6C, and then encapsulated in chitosan nanoparticles (CNP) prepared by ionic cross linkage, designated VRIL6C-CNP. The 3-week old mice were injected, respectively, with VRIL6C-CNP, VRIL6-CNP, CpG-CNP and VR1020-CNP to detect the changes of immunity. At 28 days post inoculation, the mice were challenged with virulent hemolytic serotype 2 Streptococcus to test their resistance against infection. The results showed that there was a significant increase in immunoglobulins and interleukins in mice receiving VRIL6C-CNP compared with the control groups, as well as an increase in the lymphocytes and monocytes in the inoculated mice, so that the immunity was remarkably improved in the VRIL6C-CNP group. The challenge provoked stronger immunity and protection against infection in the VRIL6C-CNP group than in the control mice that manifested severe symptoms and lesions. This suggests that VRIL6C-CNP could remarkably enhance the nonspecific immunity of mice, and facilitate the development of an effective immunopotentiator to promote the resistance of the animals against infection.

Evaluation of different culture conditions for high-level soluble expression of human cyclin A2 with pET vector in BL21 (DE3) and spectroscopic characterization of its inclusion body structure.

In this paper, we evaluated various parameters of culture condition affecting high-level soluble expression of human cyclin A(2) in Escherichia coli BL21(DE3), and demonstrated that the highest protein yield was obtained using TB(no glycerol)+0.5% glucose medium at 25 degrees C. By single immobilized metal ion affinity chromatography, we got highly purified human cyclin A(2) with a yield ranged from 20 to 30 mg/L. By amyloid-diagnostic dye ThT binding and Fourier transform infrared spectroscopy, we observed a significant decrease in alpha-helix content and an increase in beta-sheet structure in cyclin A(2) inclusion body in comparison to its native protein, and confirmed the resemblance of the internal organization of cyclin A(2) inclusion body and amyloid fibrils.

Porcine interleukin-2 gene encapsulated in chitosan nanoparticles enhances immune response of mice to piglet paratyphoid vaccine.

Interleukin-2 (IL-2) is vital to elicit and amplify the cellular and humoral immune responses to foreign antigens, which is extensively utilized in the control of infectious disease and treatment of various cancers. Porcine and murine IL-2 genes were, respectively, subcloned into VR1020, designated as VPIL-2 and VMIL-2, and then encapsulated in chitosan nanoparticles (CNP) prepared by ionic linkage. The BALB/c mice were intramuscularly co-administrated with chitosan-IL-2 nanoparticles (CNP-IL2) and paratyphoid vaccine to test the adjuvant effect of CNP-IL2. On day 35, the immunized mice were orally challenged with virulent Salmonella. The content of IgG, IgA, IgM, IL-2, IL-4, IL-6 and specific antibody titer as well as the number of immunocompetent cells were systematically analyzed in the vaccinated mice. The results revealed that the levels of immunoglobulins, cytokines, the specific antibodies, together with the numbers of lymphocytes significantly increased in vaccinated mice inoculated with CNP-VPIL2 in contrast with those with naked IL-2 plasmids and blank plasmids. The CNP-VPIL2 immunized mice exhibited higher humoral and cellular immune responses, less severe clinical signs and lesions of disease caused by the bacteria than the other groups after challenge. These findings suggest that CNP-VPIL2 has a significant enhancement effect on immune responses of mice, which results in better immunoprotection against Salmonella infection, indicating that CNP-VPIL2 could be employed as an effective immunoadjuvant to elevate immunity of animals to conventional vaccines.

Regulating effects of novel CpG chitosan-nanoparticles on immune responses of mice to porcine paratyphoid vaccines.

OBJECTIVE: To study the regulating effects of a novel CpG oligodeoxynuleotide and the synergistic effect of chitosan-nanoparticles (CNP) with CpG on immune responses of mice, which were used to develop a novel immunoadjuvant to boost immune response to conventional vaccines. METHODS: A novel CpG ODN containing 11 CpG motifs was synthesized and its bioactivities to stimulate the proliferation of lymphocytes of pig in vitro were detected. Then it was entrapped with CNP prepared in our laboratory by the method of ionic cross linkage, and immunized Kunming mice were co-inoculated with paratyphoid vaccine. The peripheral blood was collected weekly from the tail vein of inoculated mice to detect the contents of IgG, IgA, IgM, and specific antibody against salmonella as well as the levels of interleukin-2 (IL2), IL-4, and IL-6 by SABC-ELISA assay. The numbers of leucocytes, monocytes, granuloytes, and lymphocytes were calculated separately using the routine method. The experimental mice were orally challenged with virulent salmonella 35 days after inoculation. RESULTS: This CpG ODN could remarkably provoke the proliferation of lymphocytes of pig in vitro in contrast with the control (P < 0.05). Compared with those of the control, immunoglobulins, including IgG, IgA, IgM, and specific antibodies to paratyphoid vaccine, increased significantly in sera from the CpG or CpG-CNP-vaccinated mice (P < 0.05). IL-2, IL-4, and IL-6 increased remarkably in sera from immunized mice (P < 0.05). The leucocytes, monocytes, granuloytes, and lymphocytes of the mice immunized with CpG or CpG-CNP were also increased in number (P < 0.05). After the challenge, these immunity values were elevated in the mice vaccinated with CpG or CpG-CNP. The immunized mice all survived, while the control mice fell ill with evident lesions with diffuse hemorrhage in stomach, small intestine, and peritoneum. CONCLUSIONS: CpG ODN entrapped with CNP is a promising effective immunoadjuvant for vaccination, which promotes humoral and cellular immune responses, enhances immunity and resistance against salmonella by co-administration with paratyphoid vaccine.

A novel chitosan CpG nanoparticle regulates cellular and humoral immunity of mice.

OBJECTIVE: To develop a safe and novel immunoadjuvant to enhance the immunity and resistance of animals against E. coli infection. METHODS: An 88-base immunostimulatory oligodeoxynuleotide containing eleven CpG motifs (CpG ODN) was synthesized and amplified by PCR. The chitosan nanoparticle (CNP) was prepared by ion linking method to entrap the CpG ODN that significantly promotes the proliferation of lymphocytes of pig in vitro. Then the CpG-CNP was inoculated into 21-day old Kunming mice, which were orally challenged with virulent K88/K99 E. Coli 35 days after inoculation. Blood was collected from the tail vein of mice on days 0, 7, 14, 21, 28, 35, 42, and 49 after inoculation to detect the changes and content of immunoglobulins, cytokines and immune cells by ELISA, such as IgG, IgA, IgM, IL-2, IL-4, and IL-6. RESULTS: The CpG provoked remarkable proliferation of lymphocytes of pig in vitro in comparison with that of control group (P < 0.05). The inoculation with CpG-CNP significantly raised the content of IgG, IgM, and IgA in the sera of immunized mice (P < 0.05). The levels of IL-2, IL-4, and IL-6 in the mice significantly increased in comparison with those in controls (P < 0.05), so was the number of white blood cells and lymphocytes in immunized mice. The humoral and cellular immunities were significantly enhanced in immunized mice, which resisted the infection of E. coli and survived, while the control mice manifested evident symptoms and lesions of infection. CONCLUSIONS: CpG-CNP can significantly promote cellular and humoral immunity and resistance of mice against E. coil infection, and can be utilized as an effective adjuvant to improve the immunoprotection and resistance of porcine against infectious disease.

Molecular cloning and expression of IL2 cDNA from the Tibet pig.

Interleukin-2 is a vital cytokine secreted by activated T lymphocytes, and plays important role in the regulation of cellular and humoral immunity of animals. In our experiment, IL2 cDNA of the Tibet Pig was first cloned by RT-PCR from ConA-stimulated lymphocytes in the blood and subcloned into pMD-18 T vector, which then was identified with endonuclease restriction. The sequencing result showed that Tibet pig IL-2 (TPIL-2) cDNA was 503 bp long (ORF was 465 bp) (Genbank accession number: AY 294018). The recombinant prokaryotic and eukaryotic expression plasmids of the cDNA were then constructed to analyse the ability to stimulate the proliferation of porcine lymphocytes in vitro. The recombinant porcine IL-2 expressed in the prokaryotic cells was found to be of 43 kDa molecular mass, which was consistent with a 17.4 kDa protein deduced from the IL-2 cDNA sequence (glutathione S-transferase molecular mass is 26 kDa); the recombinant protein in eukaryotic cells was confirmed by use of specific rabbit anti-porcine IL-2 serum in an ELISA. The bioactivity of TPIL-2 was detected through MTT colorimetry by stimulating the proliferation of pig ConA-stimulated blasts in vitro. The results indicate that the TPIL-2 significantly promoted the proliferation of ConA-stimulated blasts of pig. This confirms that IL-2 cDNA of the Tibet pig was successfully cloned and expressed in prokaryotic and eukaryotic cells, which lays the foundation for the the preparation of specific recombinant IL-2 protein and development of novel immune adjuvants to raise the immunity of pigs against various infectious pathogens and increase the immunoprotective efficacy of vaccines.

[Effects of entrapment of murine interleukin-2 gene with chitosan nanoparticles on expression of mIL-2 gene and on regulation of immune response in mice].

The experiment was conducted to prepare chitosan nanoparticles (CNP), to entrap VRMIL-2 with CNP, the eukaryotic VR1020 expression plasmid containing murine IL-2 gene (mlL-2), and to investigate the expression in vivo and the regulatory effect of mIL-2 on immune-response and immuno-protection in mice inoculated muscularly with CNP entrapped VMIL-2 at 21 days old. The results showed that IgG, IgM and IgA contents increased to different degrees in the sera from the inoculated mice, which were remarkably higher than those of the controls inoculated VR1020 packed with CNP (P<0.05); so were the IL-2, IL-4 and IL-6 contents in the sera of the immunized mice. The number of white blood cells and lymphocytes significantly increased respectively in the vaccinated mice, compared with those of controls. These mice were orally challenged with virulent E. coli 35 days post-inoculation, and all the immune responses were significantly higher than those of the control except the number of neutrophils. The mice inoculated with VRMIL-2 survived healthily, while the mice of control group were ill with the evident lesions. Although there are no remarkable differences between the cellular and humoral immune indexes of mice inoculated with CNP-VRMIL-2 and nude VRMIL-2 (P>0.05), the dosage of CNP-VRMIL-2 is only one fifth of the VRMIL-2. These indicated that entrapment of mIL-2 gene with chitosan nanoparticles could remarkably enhance the expression of mIL-2 in vivo, and significantly raise the levels of cellular and humoral immune, and increase the resistance of mice against E. coli infection. The results suggested that chitosan nanoparticles and IL-2 gene could be used as an effective immunoenhancer to increase the immunity of animals against infection.