Objective To provide experimental evidences for developing a safe and effective re- combinant fowlpox virus which can prevent the infection of HIV-2.Methods A fowlpox virus(FPV) transferring vector was constructed by inserting HIV-2 gag gene to the downstream of a synthetic complex promoter ATI-p7.5?20 of vector pUTA2.Transfection was then carried out,and recombi- nant FPV(rFPV)was screened by 5'-bromo-deoxyuridine(BrdU),genome PCR and western blot detection.Balb/c mice were immunized with rFPV by muscular injection.Anti-HIV-2 antibody, CD4~+ and CD8~+ T-cell count and specific target-killing activity of spleen CTL in immunized mice were analyzed by ELISA,FACS and LDH release assay,respectively.Results A transferring vector pA- gag was constructed and confirmed by amplifying a fragment of 766 bp from the rFPV genome.Mean- while,HIV-2 multi-antibody-specific protein blot(55 000)was detected from the recombinant virus and the HIV-2 specific antibody was detected from the immunized Balb/c mice.HIV-2 specific target- killing activity of spleen CTL was observed in immunized mice.Conclusion A recombinant fowlpox virus expressing HIV-2 structural protein Gag has been obtained,and it can stimulate HIV-2-specific eelluar and humoral immune reactions in mice.

To investigate the chemical constituents of the whole plants of Bidens bipinnata, the separation and purification of constituents were performed by chromatography on macroporous resin, silica gel, MCI and Sephadex LH-20. Their structures were elucidated by spectroscopic data as quercetin (1), quercetin-3-0-alpha-L-rhamnoside (2), keampferol-3-O-beta-D-glucopyranoside (3), keampferol-3-O-alpha-L-rhamnoside (4), 3', 5-dyhydroxy-3, 6, 4'-trimethoxyl -7-O-beta-D-glucopyranoside flavonoid (5), 7, 8, 3', 4'-tetraflavanone(6), (2S)- and (2R)-isookanin-7-O-beta-D- glucopyranoside (7a/7b), (2S)- and (2R)-3'-methoxy-isookanin-8-O-beta-D-glucopyranoside (8a/8b), 6, 7, 3', 4'-tetrahydroxyaurone(9), maritimetin (10), esculetin (11), 3-O-caffeoyl-2-methyl-d-erythrono-1, 4-lactone (12), (7S, 8R) balanophonin-4-O-beta-D-glucopyranoside (13), eugenyl-O-beta-apiofuranosyl-( 1"-6') -O-beta-glucopyranoside (14), and (+)-syringaresinol-4'-O-beta-D-glucopyranoside (15). Compounds 8, 13, 14, and 15 were isolated from this genus for the first time. Compounds 1 and 6 were potent inhibitors against HSC-T6 cells in vitro and compounds 1, 2, 6, and 7 were capable of decreasing the inflammatory cytokine production of macrophage cells in vitro.
Bidens ; chemistry ; Drugs, Chinese Herbal ; chemistry ; Molecular Structure ; Spectrometry, Mass, Electrospray Ionization

OBJECTIVETo discuss mass spectrum characterization of five valepotriates including 'monoene' type (didrovaltrate), 'diene' type (valtrate, acevaltrate) and 'four-olefinic' type (baldrinal and homobaldrinal) by electrospray ionization tandem mass spectrometry (ESI-MS(n)).

METHODThis study was carried out on the basis of electrospray ionization tandem mass spectrometric method and analysis of multistage fragments.

RESULTThe fragmentation patterns and structural assignment of 'monoene' type, 'diene' type and 'four-olefinic' type valepotriates in ESI-MSn under positive mode were summarized.

CONCLUSIONThe compounds have a strong pyrolysis rules and it can provide reference date for valepotriates in rapid structural identification, quantitative analysis and pharmacokinetic study.

Drugs, Chinese Herbal ; chemistry ; Iridoids ; chemistry ; Spectrometry, Mass, Electrospray Ionization ; Tandem Mass Spectrometry

To screening out Chinese vaccine candidate against HIV-1, Chinese vaccine strain 282E4 of fowlpox virus was used as the vector to construct the recombinant fowlpox virus (rFPV) coexpressing gp120 of Chinese HIV-1 strain and IL-18, and the recombinant virus was indentified by PCR and Western blot. The specific DNA fragment could be amplified by PCR from the genome of rFPV. Western blot analysis showed that gp120 and IL-18 could be expressed not only in chicken embryo fibroblast (CEF) cells infected by rFPV, but also in mammalian cells infected by rFPV. After the recombinant fowlpox virus was inoculated into BALB/c mice, the spleen specific CTL activities and serum antibodies in the immunized mice were detected, which demonstrated that the rFPV had good immunogenicity and could induce BALB/c mice to produce specific humoral and cellular immunity. IL-18 palyed the role of immunoadjuvant. The study lays the basis on the preparation of genetic engineering live vector vaccine against HIV-1.
AIDS Vaccines ; immunology ; Adjuvants, Immunologic ; Animals ; Antibodies, Viral ; immunology ; Fowlpox virus ; genetics ; immunology ; metabolism ; Gene Transfer Techniques ; Genetic Vectors ; HIV Envelope Protein gp120 ; biosynthesis ; genetics ; immunology ; HIV-1 ; genetics ; immunology ; Humans ; Immunization ; Interleukin-18 ; biosynthesis ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Vaccines, DNA ; immunology

BACKGROUNDThe results of clinical trials of rapamycin-eluting stents reduce restenosis have been quite promising. The main purpose of this study was to characterize the in vivo pharmacokinetics of high dose rapamycin (Rapa)-eluting stents in a miniswine coronary model.

METHODSTen miniswines underwent placement of 18 high dose Rapa-eluting stents in the left anterior descending and right coronary arteries. At the planned times of the 1.5th, 12th, 24th hour, 3th, 7th and 28th day, the animals (n = 1, 1, 2, 2, 2, and 2, respectively) were euthanized after completion of coronary angiography. Blood samples were obtained at 0, 10, 20, 30 minutes; 1, 2, 6, 24 hours; and 3, 7, 28 days to determine systemic Rapa levels. Rapa levels in whole blood, arterial wall, heart, renal and liver tissues were determined by high-performance liquid chromatography/mass spectroscopy.

RESULTSPeak whole blood concentration (Cmax), time to peak concentration (tmax), elimination half-life (t1/2beta), area under the curve (AUC), and apparent systemic clearance (Cl/F) were (10.91 +/- 1.28) ng/ml, (2.0 +/- 0.2) hours, (7.25 +/- 0.63) hours, (1.15 +/- 0.11) ng x h x ml(-1), and (180 +/- 12) ml x h(-1) x kg(-1), respectively. More than 95% Rapa detected is localized in the coronary artery surrounding the stent and heart.

CONCLUSIONStent-based delivery of Rapa via a copolymer stent is feasible and safe. This strategy holds promise for the prevention of stent restenosis.

Animals ; Chromatography, High Pressure Liquid ; Coronary Restenosis ; prevention & control ; Male ; Mass Spectrometry ; Sirolimus ; administration & dosage ; pharmacokinetics ; Stents ; Swine ; Swine, Miniature ; Tissue Distribution

Anti-HIV-1 gp120 single chain antibody(scFv) gene and staphylococcus extoxin A(SEA) gene were inserted into vector pPIC9K. The recombinant plasmid was integrated into Pichia pastoris by electroporation. High level expression was performed by determining the Muts phenotype and screening muti-copy integrants. The recombinant protein was about 57kD and the production was 50.1 mg/L. It was shown that the two kinds of protein affected the conformation of each other by antibody affinity assay, but the recombinant targeting toxins could highly mediate CTLs to kill HIV-1 target cells.
Enterotoxins ; genetics ; Genetic Vectors ; HIV Envelope Protein gp120 ; biosynthesis ; genetics ; immunology ; HIV-1 ; genetics ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; immunology

Breast Neoplasms/surgery* ; Carcinoma in Situ ; Carcinoma, Ductal, Breast ; Carcinoma, Intraductal, Noninfiltrating/surgery* ; China ; Female ; Humans ; Mastectomy

OBJECTIVETo investigate the feasibility of monitoring therapeutic effect of adenovirus vector containing IL12-IRES-CKb gene on a rabbit VX2 liver tumor model by using phosphorous-31 magnetic resonance spectroscopy (31P MRS).

METHODSA total of 18 healthy New Zealand White rabbits were used to generate animal models by implanting VX2 tumor chips into livers through laparotomy. Tumor-bearing animals were randomly divided into three groups and were injected with AdCMVIL12-IRES-CKb, AdCMV-Empty and saline respectively via ear veins. 31P MRS scan was performed after animals were fed with creatine solution for five days. Animals were euthanized thereafter and tumors were removed for pathological examination, immunohistochemistry (IHC) staining and protein analysis (Western blot).

RESULTSThe intrahepatic and seral expressions of creatine kinase (CKb) and IL-12 were detected only in AdCMVIL12-IRES-CKb group. Tumor diameters pre- and post- treatment in three groups were 1.63+/-0.04 vs 1.62+/-0.03 in AdCMVIL12-IRES-CKb group (P = 0.229), 1.59+/-0.05 vs 1.84+/-0.11 in AdCMV-Empty group (P = 0.003) and 1.60+/-0.02 vs 2.07+/-0.12 in saline group (P = 0.001), respectively. Pcr Changes between pre- and post- treatment among the three groups were compared (F = 6.235, P value is less than 0.05). PCr increased significantly in AdCMVIL12-IRES-CKb group as compared to AdCMV-Empty (P = 0.004) and saline group (P = 0.049), whereas no change found between AdCMV-Empty and saline group (P = 0.153).

CONCLUSION31P MRS, an effective and non-invasive functional imaging method, can be used to monitor the therapeutic effect of adenovirus vector containing IL12-IRES-CKb gene on rabbit VX2 liver tumor model through detecting metabolic product of imaging reporter gene CKb (pCr).

Adenoviridae ; genetics ; Animals ; Creatine Kinase ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Interleukin-12 ; genetics ; Liver Neoplasms, Experimental ; genetics ; pathology ; Magnetic Resonance Spectroscopy ; Rabbits

OBJECTIVETo construct the recombinant fowlpox virus (rFPV) coexpressing HIV-1 gag-gp120 and hIL-6.

METHODSThe recombinant expressing plasmid pUTA-GE-IL6 was successfully constructed by inserting gag-gp120 gene and hIL-6 gene into the downstream of the combined promoter ATI-p7.5 and p7.5 tandem promoter respectively. After transfecting the plasmid into chicken embryonic fibroblast (CEF) cells preinfected with FPV 282E4 strain and selecting the recombinant virus under the pressure of BUdR. The recombinant virus was analyzed by nucleic acid probe hybridization and immunoblotting. In addition, the formation of virus-like particle and the expression of interested proteins in the recombinant virus-infected p815 cells were observed, and the immunogenicity of the recombinant virus was also analyzed.

RESULTSThere was colorable dot for the positive recombinant virus, immunoblotting analysis showed that the recombinant virus could expressed both gag-gp120 and IL-6. Virus-like particles (VLP) were formed in virus-infected cells, and the interested proteins could be expressed in mammalian cells infected by the recombinant virus. The immunity index from the immunized mice showed that the recombinant virus had good immunogenicity.

CONCLUSIONThe recombinant fowlpox virus coexpressing gag-gp120 and IL-6 was successfully constructed, which may provide basis for the preparation of live vector genetic engineering vaccine and macromolecule particle vaccine against HIV-1.

Animals ; Antibodies, Viral ; blood ; Blotting, Western ; Cells, Cultured ; Chick Embryo ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Fibroblasts ; cytology ; metabolism ; ultrastructure ; Fowlpox ; blood ; immunology ; virology ; Fowlpox virus ; genetics ; immunology ; Gene Products, gag ; genetics ; metabolism ; Genetic Vectors ; genetics ; HIV Envelope Protein gp120 ; genetics ; metabolism ; HIV-1 ; genetics ; metabolism ; Immunization ; methods ; Interleukin-6 ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Microscopy, Electron ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Transfection ; Viral Vaccines ; genetics ; immunology ; metabolism

OBJECTIVETo evaluate the prevalence and distribution of C-kit, NPM1 and FLT3 gene mutations in patients with acute myeloid leukemia (AML), and to analyze the relationship between the gene mutations and their prognosis.

METHODSMutations in exon 8 and 17 of C-kit gene, exon 12 of NPM1 gene, exon 20 of FLT3-TKD gene, and exon 14/15 of FLT3-ITD gene were detected by direct sequencing. Clinical data was collected and followed up if the patient had accepted treatment in our hospital.

RESULTSAmong the 656 AML patients, mutations in C-kit exon 8 were found in 6 patients (0.9%), C-kit exon 17 in 33 (5.0%), NPM1 in 169 (25.8%), FLT3-TKD in 46 (7.1%), and FLT3-ITD in 178 (27.1%). Six subtypes of mutations were detected in C-kit exon 8, 8 in C-kit exon 17, 11 in FLT3-TKD, 15 in NPM1, of which 5 were not reported before. C-kit exon 17 mutations were more frequently detected in patients with t(8;21) and exon 8 in patients with inv(16) cytogenetic abnormality. No other gene mutations except FLT3 were detected in M(3) patients. NPM1 and ITD mutations were often detected in individuals with normal cytogenetics or M(5) and M(1) of FAB classification, and accompanied with high white blood cell counts in peripheral blood, high blast counts in bone marrow and low CD34 expression. The older the patients were when diagnosed, the more gene mutations and the higher white blood cell count were detected. More mutations were found in individuals with normal karyotype than that with other karyotypes. It appeared that FLT3-ITD was significantly associated with shorter overall survival (OS) (P = 0.004), NPM1 was not significantly associated with OS, but NPM1(+)/ITD(-) patients had the longest OS.

CONCLUSIONSOur results showed that the mutation types and amounts had particular distribution in MICM subtypes, and were associated with white blood cell counts in peripheral blood, blast counts in bone marrow and prognosis. Especially for patients with normal karyotype, the genetic mutations could be new molecule marker.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Asian Continental Ancestry Group ; genetics ; DNA Mutational Analysis ; Female ; Humans ; Karyotyping ; Leukemia, Myeloid, Acute ; diagnosis ; genetics ; Male ; Middle Aged ; Mutation ; Nuclear Proteins ; genetics ; Prognosis ; Proto-Oncogene Proteins c-kit ; genetics ; Young Adult ; fms-Like Tyrosine Kinase 3 ; genetics