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Biomedical subjects

Rong Gao

Publications and source records attributed to Rong Gao.

At least 19 recordsLinked to original sources
PubMed2006

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.

Adjuvants, Immunologic↗
PubMed2006

Synthesis and biological evaluation of 10,11-methylenedioxy-14-azacamptothecin.

[reaction: see text] 10,11-Methylenedioxy-14-azacamptothecin, a potent analogue of the antitumor agent camptothecin (CPT), has been prepared via a key condensation between AB and DE ring precursors. The biological testing of this compound validated a strategy for modulation of the off-rate of camptothecin analogues from the topoisomerase-DNA-CPT ternary complex via structural modification.

Antineoplastic Agents, Phytogenic↗
PubMed2006

Gradient high-performance liquid chromatography for the simultaneous determination of chlorogenic acid and baicalin in plasma and its application in the study of pharmacokinetics in rats.

A novel HPLC-UV method was developed for the simultaneous determination of two major active components in Yinhuang injection, chlorogenic acid and baicalin, in rat plasma. Extracted from the plasma samples with methanol-acetonitrile (3:1, v/v), the two compounds were successfully separated using a C18 column with a gradient elution composed of 15 and 54% methanol-acetonitrile (1:1, v/v) in 0.2% (v/v) phosphoric acid water solution (pH 2.0). The flow-rate was set at 1 ml min(-1) and the eluent was detected at 327 nm for chlorogenic acid, 278 nm for baicalin. Puerarin and rutin were used as the internal standards for chlorogenic acid and baicalin, respectively. The method was linear over the range of 0.388-12.4 microg ml(-1), 0.485-124 microg ml(-1) for chlorogenic acid and baicalin, respectively. The correlation coefficient for each analyte was above 0.998. The intra-day and inter-day precisions were better than 7 and 9%, with the relative error ranging from -9.5 to 7.3% and from -4.2 to 1.8%. The limit of detection (LOD) and the limit of quantification (LOQ) for chlorogenic acid and baicalin in plasma were 0.194, 0.122, 0.388 and 0.485 microg ml(-1), respectively. This assay has been successfully applied in the pharmacokinetic study of chlorogenic acid and baicalin in vivo through intravenous administration of Yinhuang injection to rats.

Animals↗
PubMed2006

Effects of modification of the active site tyrosine of human DNA topoisomerase I.

The human topoisomerase I-mediated DNA relaxation reaction was studied following modification of the enzyme at the active site tyrosine (position 723). A series of unnatural tyrosine analogues was incorporated into the active site of human topoisomerase I by utilizing misacylated suppressor tRNAs in an in vitro protein synthesizing system. The relaxation activities of the modified human topoisomerase I analogues having varied steric, electronic, and stereochemical features were all greatly diminished relative to that of the wild type. It was found that modifications involving replacement of the nucleophilic tyrosine OH group with NH2, SH, or I groups eliminated DNA relaxation activity, as did changing the orientation of the nucleophilic tyrosine OH group. Only tyrosine analogues having the phenolic OH group in the normal position with respect to the protein backbone were active; the relative activities could be rationalized in chemical terms on the basis of the H-bonding and the electronic effects of the substituents attached to the meta position of the aromatic ring. In addition, the poisoning of one of the modified human topoisomerase I analogues, as part of covalent binary complexes with DNA, by CPT and 20-thio CPT was evaluated.

Binding Sites↗
PubMed2006

Constitutive activation of two-component response regulators: characterization of VirG activation in Agrobacterium tumefaciens.

Response regulators are the ultimate modulators in two-component signal transduction pathways. The N-terminal receiver domains generally accept phosphates from cognate histidine kinases to control output. VirG for example, the response regulator of the VirA/VirG two-component system in Agrobacterium tumefaciens, mediates the expression of virulence genes in response to plant host signals. Response regulators have a highly conserved structure and share a similar conformational activation upon phosphorylation, yet the sequence and structural features that determine or perturb the cooperative activation events are ill defined. Here we use VirG and the unique features of the Agrobacterium system to extend our understanding of the response regulator activation. Two previously isolated constitutive VirG mutants, VirGN54D and VirGI77V/D52E, provide the foundation for our studies. In vivo phosphorylation patterns establish that VirGN54D is able to accumulate phosphates from small-molecule phosphate donors, such as acetyl phosphate, while the VirGI77V/D52E allele carries conformational changes mimicking the active conformation. Further structural alterations on these two alleles begin to reveal the changes necessary for response regulator activation.

Agrobacterium tumefaciens↗
PubMed2006

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.

Adjuvants, Immunologic↗
PubMed2006

[Effects of flunarizine on T-type calcium channels in mouse spermatogenic cells].

OBJECTIVE: To investigate the effects of Flunarizine (Flu) on T-type calcium currents (ICaT) in spermatogenic cells. METHODS: Ca2+ currents were obtained in acutely dissociated mouse spermatogenic cells using whole-cell patch clamp technique and the effects of Flu on ICaT were observed. RESULTS: Flu of 0.1, 1, 10, 100 micromol/L inhibited ICaT in mouse spermatogenic cells significantly with the K50 value of 0.289 micromol/L (P < 0.05). With the holding potential at -90 mV and stimulating potential at -30 mV, the inhibition rates of Flu were (23.34 +/- 2.76)%, (46.04 +/- 3.52)%, (62.52 +/- 3.70)% and (73.52 +/- 3.12)%, respectively. CONCLUSION: Flu has significant inhibitory effects on ICaT in mouse spermatogenic cells, with concentration dependence. Ca(v)3.2 is the main contributor to T-type Ca2+ currents in spermatogenic cells.

Animals↗
PubMed2006

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.

Adjuvants, Immunologic↗
PubMed2005

Fenvalerate modifies T-type Ca2+ channels in mouse spermatogenic cells.

Prior to fertilization sperm must undergo the acrosome reaction that is initiated by opening of T-type Ca(2+) channel. Hence, T-type Ca(2+) channels could be a potential target for agents affecting the acrosome reaction. Our previous data have suggested that fenvalerate, a type II pyrethroid, inhibited the acrosome reaction in mice. To elucidate its potential mechanism we investigated fenvalerate's effect on T-type Ca(2+) channels in mouse pachytene spermatocytes using a whole-cell patch clamp technique. Fenvalerate significantly inhibited T-type Ca(2+) currents in a concentration-dependent manner. The maximal inhibitory concentration was 1 microM. The inhibitory effect of fenvalerate was slow and irreversible after washout of the drug. The curves of activation and inactivation simultaneously shifted to hyperpolarization, about 14 mV, suggesting the open time of Ca(2+) channel was unchanged. Voltage-dependent gating of Ca(2+) channel indicated a change in permeability to ions that contributed to fenvalerate's inhibition on Ca(2+) current. Taken together with our previous findings, these data suggest that the changes of T-type Ca(2+) currents contribute to the suppression of acrosome reaction and fertilizing ability caused by fenvalerate.

Animals↗
PubMed2005

Analogues of vaccinia virus DNA topoisomerase I modified at the active site tyrosine.

The mechanism of type IB topoisomerase-mediated DNA relaxation was studied by modification of vaccinia topoisomerase I at the active site tyrosine (position 274) with several tyrosine analogues. These analogues had varied steric, electronic, and stereochemical features to permit assessment of those structural elements required to support topoisomerase function. Eleven tyrosine analogues were successfully incorporated into the active site of vaccinia topoisomerase I. It was found that only tyrosine analogues having the phenolic -OH group in the normal position relative to the protein backbone were active. Modifications that replaced the nucleophilic tyrosine OH (pKa approximately 10.0) group with NH2 (pKa 4.6), SH (pKa approximately 7.0), or I groups or that changed the orientation of the nucleophilic OH group essentially eliminated topoisomerase I function. For the active analogues, the electronic effects and H-bonding characteristics of substituents in the meta-position of the aromatic ring may be important in modulating topoisomerase I function. The pH profile for the functional analogues revealed a small shift toward lower pH when compared with wild-type topoisomerase I.

Amino Acid Sequence↗
PubMed2005

Synthesis of 14-azacamptothecin, a water-soluble topoisomerase I poison.

14-Azacamptothecin, a potent, water-soluble analogue of the antitumor agent camptothecin, has been prepared by a convergent synthesis. The key condensation of the AB and DE rings with concomitant formation of ring C of 14-aza CPT was carried out in two stages, the latter of which involved a radical cyclization strategy. [structure: see text]

Antineoplastic Agents↗
PubMed2005

On the role of E-ring oxygen atoms in the binding of camptothecin to the topoisomerase I-DNA covalent binary complex.

A recent X-ray crystallographic analysis of the binding of a water soluble camptothecin analogue to the human topoisomerase I-DNA covalent binary complex has suggested the existence of some novel features in the way that camptothecin is bound to the binary complex. Four additional models based on chemical and biochemical data have also been proposed. Presently we describe S-containing analogues of camptothecin prepared on the basis of these models, and report their ability to form stable ternary complexes with human topoisomerase I, and to mediate cytotoxicity at the locus of topoisomerase I. The results indicate that replacement of the 20-OH group of CPT with a SH functionality results in diminution of the potency of CPT as a topoisomerase I poison, while replacement of the O atoms at positions 20 and 21 with S atoms results in essentially complete loss of topoisomerase I inhibitory activity.

Autoradiography↗
PubMed2005

14-azacamptothecin: a potent water-soluble topoisomerase I poison.

On the basis of an analysis of luotonin A and its D-ring deaza analogue as topoisomerase I poisons and topoisomerase I-dependent cytotoxic agents, a novel analogue of the structurally related antitumor antibiotic camptothecin (CPT) was prepared. 14-Azacamptothecin was found to have much greater aqueous solubility than CPT, to inhibit topoisomerase I-mediated DNA relaxation more efficiently than CPT, and to stabilize the covalent binary complex to almost the same extent. 14-Aza CPT was found to be slightly less active than CPT in mediating cytotoxicity toward yeast expressing human topoisomerase I, possibly as a consequence of its greater off-rate from the CPT-topoisomerase I-DNA ternary complex.

Camptothecin↗
PubMed2005

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.

Animals↗
PubMed2005

Structure and function comparison of Micropechis ikaheka snake venom phospholipase A2 isoenzymes.

Comparison of the crystal structures of three Micropechis ikaheka phospholipase A2 isoenzymes (MiPLA2, MiPLA3 and MiPLA4, which exhibit different levels of pharmacological effects) shows that their C-terminus (residues 110-124) is the most variable. M-Type receptor binding affinity of the isoenzymes has also been investigated and MiPLA4 binds to the rabbit M-type receptor with high affinity. Examination of surface charges of the isoenzymes reveals a trend of increase in positive charges with potency. The isoenzymes are shown to oligomerize in a concentration-dependent manner in a semi-denaturing gel. The C-termini of the medium (MiPLA4) and highly potent (MiPLA2) isoenzyme molecules cluster together, forming a highly exposed area. A BLAST search using the sequence of the most potent MiPLA2 results in high similarity to Staphylococcus aureus clotting factor A and cadherin 11. This might explain the myotoxicity, anticoagulant and hemoglobinuria effects of MiPLA2s.

Amino Acid Sequence↗
PubMed2005

Environmental pH sensing: resolving the VirA/VirG two-component system inputs for Agrobacterium pathogenesis.

Agrobacterium tumefaciens stands as one of biotechnology's greatest successes, with all plant genetic engineering building on the strategies of this pathogen. By integrating responses to external pHs, phenols, and monosaccharides, this organism mobilizes oncogenic elements to efficiently transform most dicotyledonous plants. We now show that the complex signaling network used to regulate lateral gene transfer can be resolved as individual signaling modules. While pH and sugar perception are coupled through a common pathway, requiring both low pH and sugar for maximal virulence gene expression, various VirA and ChvE alleles can decouple pH and monosaccharide perception. This VirA and ChvE system may represent a common mechanism that underpins external pH perception in prokaryotes, and the use of these simple genetic elements may now be extended to research on specific responses to changes in environmental pH.

Agrobacterium tumefaciens↗
PubMed2005

[Detection of autoimmune parameter of SARS patients].

OBJECTIVE: To explore whether autoimmune phenomena exist in SARS patients, and to seek for unusual autoimmune antibodies in SARS patients. METHODS: Autoantibodies against cell nuclei (ANA), autoantibodies against smooth muscles (SMA), autoantibodies against parietal cells (PCA), autoantibodies against heart cells (HRA) were detected by using immunofluorescence, and autoantibodies against live-kidney microsomes (LKM) and anti-M2 antibodies were detected by ELISA in sera taken from 27 SARS patients and 18 healthy controls. Immunofluorescence was used to localize the targets antigens in slides with biochips of lung (monkey) of SARS associated antibodies. RESULTS: ANA, AMA, LKM and SMA were found positive in 3, 1, 1, and 1 of the 27 SARS sera. In 18 healthy control sera, one ANA and one AMA were positive. Statistical analysis showed that there were no difference between two groups in every item detected. Twenty-six of 27 SARS patients and 5 of 18 healthy controls had strongly stained columnar epithelia of the bronchiole, especially the lumen border of the epithelia?the difference between two groups was significant. CONCLUSION: No antibodies against organs but lung were found in SARS patients. There are auto antibodies against lung tissues in sera of SARS patients.

Adolescent↗