Aim According to various target sites of HLA-E mRNA,to design and synthesize 3 pieces of HLA-E siRNA chain,to compare quantitatively their efficiency of silencing gene in BEL-7402 with HLA-E(+)in order to select the dominant siRNA.Methods The hepatocarcinomal BEL-7402 cells,induced by 5×10~5 IU·L~(-1) IFN-γ,expressed HLA-E(+) and was pured by flow cytometry selecting as target cells for research.3 pieces of specific siRNA(A,B,C group)were designed and chemically synthesized,then the concentration of which(0.1 mmol·L~(-1))was respectively transfected through Lipofectamin 2000 into target cells.After 48 h,the gene silent effect on HLA-E gene in A,B and C groups was quantitatively observed by cytoimmunofluorence,flow cytometery,Western blot and real-time PCR,as well as on NK cytotoxicity to target cells tested by NK killing rate.Results Compared with those of control or non-specific siRNA group,HLA-E antigen,protein product,HLA-E mRNA and HLA-E molecule on cell surface were statistically down-regulated in A,B,and C group(P<0.01),whose were silenced more (above 90%) in B or C group than in A group (P<0.01).The NK killing rate in A,B and C groups was dominantly improved(P<0.01),which in B or C group was higher than in A group (P<0.01).Conclusion The targeted siRNA can specifically and high-efficiently silence HLA-E expression in hepatocarcinomal cells,and may keep them from immunoescape through non-classic HLAⅠ pathway to imply new strategy for hepatocarcinomal gene-immunotherapy.

Alanine Transaminase ; metabolism ; Animals ; Liver ; drug effects ; Male ; Mice ; Plant Extracts ; pharmacology

The study aims to investigate the embryo-fetus development toxicity of the novel PPAR-δ agonist HS060098 on SD rats. The pregnant rats that were randomly divided into the solvent control group (1% hydroxypropyl methyl cellulose water solution) and HS060098 suspension groups (10, 30 and 100 mg x kg(-1) xd(-1)) were orally administered with HS060098 suspension or vehicle during the gestation of 6 -15 days (GD6-15). At termination (GD20), female rats were sacrificed. The pregnant females were evaluated by corpora lutea count, implantation sites, existence and death of embryos. Fetal sex, weight, externals, variations and malformations of viscus and skeleton were observed. The results show that there were no significant abnormality in maternal general conditions and fetal appearance as well as viscera, but in the 100 mg x kg(-1) x d(-1) group, the maternal weight gain decreased greatly (P < 0.01) and the skeletal ossification delayed remarkably (P < 0.01); in the 30 mg x kg(-1) xd(-1) group, the fatal and litter number of incompletely ossified sternebrae II was higher than those of the control group (P < 0.05); the skeletal malformations occurred in all dose groups, which indicate that the novel PPAR-δ agonist HS060098 had maternal toxicity and adversely effected fetal skeletal development under the experimental conditions.
Animals ; Bone and Bones ; drug effects ; Embryonic Development ; drug effects ; Female ; Fetal Weight ; PPAR delta ; agonists ; Pregnancy ; Rats ; Toxicity Tests

To provide accurate information on geographic distribution of crude drug Sailonggu in the plateau, we identified zokor species (Eospalax spp.) in Qinghai-Tibet Plateau using molecular methods. Based on the mitochondrial cytochrome B (cytb) gene sequences, we then extracted haplotypes from these sequences and reconstructed phylogenetic trees for the haplotypes using both maximum likelihood (ML) and Bayesian inference (BI) methods. Based on the trees, the species of each sample were determined. Five hundred and three samples from 35 populations were sequenced and their whole cytb sequences (1140 bp) were obtained. From these sequences 150 haplotypes were detected, in which, 126 were Eospalax baileyi, 20 were E. cansus, and 4 were E. smithi of the 35 populations, 28 were E. baileyi type, 5 were E. cansus type, and the remaining 2 were mixed of E. baileyi + E. cansus (DT2) and E. baileyi + E. smithi (ZN3). The results showed that, the regions around the Qinghai lake and near the upper stream of Yellow River started at Guide could be viewed as the producing area of authentic Sailonggu, and also, the cytb gene is a powerful molecular marker to determine the species of zokors as well as for the authentication of geographic distribution of Sailonggu.
Animals ; Bone and Bones ; metabolism ; Haplotypes ; Medicine, Tibetan Traditional ; Phylogeny ; Rodentia ; classification ; genetics

Carotid Artery Injuries ; therapy ; Carotid-Cavernous Sinus Fistula ; therapy ; Embolization, Therapeutic ; methods ; Humans ; Male ; Middle Aged ; Stents

PURPOSE: Interleukin (IL)-13, a Th2-type cytokine, plays a pivotal role in the pathogenesis of asthma through its direct effects on airway smooth muscles. A naturally occurring IL-13 polymorphism, R110Q, is strongly associated with increased total serum IgE levels and asthma. In the present study, we aimed to determine whether the IL-13 R110Q variant would display different biochemical properties or altered functions in comparison with wild-type (WT) IL-13 in cultured human bronchial smooth muscle cells (hBSMCs). METHODS: Culture supernatants and cell proteins were collected from cultured hBSMCs that were treated with 50 ng/mL IL-13 or IL-13 R110Q for 24 hours. Eotaxin released into hBSMC culture medium was determined by ELISA. The expression levels of the high-affinity IgE receptor (FcepsilonRI) alpha-chain, smooth muscle-specific actin alpha chain (alpha-SMA), smooth muscle myosin heavy chain (SmMHC), and calreticulin in the cells were measured on Western blots. RESULTS: Compared with WT IL-13, treatment with the IL-13 R110Q variant resulted in a significant increase in eotaxin release as well as significant, although modest, increases in the expression levels of alpha-SMA, SmMHC, calreticulin, and FcepsilonRI alpha-chain. CONCLUSIONS: The results of the present study suggenst that the IL-13 R110Q variant may enhance enhanced functional activities in hBSMCs.
Actins ; Asthma ; Calreticulin ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin E ; Interleukin-13 ; Interleukins ; Muscle, Smooth ; Myocytes, Smooth Muscle ; Myosin Heavy Chains ; Proteins

PURPOSE: Interleukin (IL)-13, a Th2-type cytokine, plays a pivotal role in the pathogenesis of asthma through its direct effects on airway smooth muscles. A naturally occurring IL-13 polymorphism, R110Q, is strongly associated with increased total serum IgE levels and asthma. In the present study, we aimed to determine whether the IL-13 R110Q variant would display different biochemical properties or altered functions in comparison with wild-type (WT) IL-13 in cultured human bronchial smooth muscle cells (hBSMCs). METHODS: Culture supernatants and cell proteins were collected from cultured hBSMCs that were treated with 50 ng/mL IL-13 or IL-13 R110Q for 24 hours. Eotaxin released into hBSMC culture medium was determined by ELISA. The expression levels of the high-affinity IgE receptor (FcepsilonRI) alpha-chain, smooth muscle-specific actin alpha chain (alpha-SMA), smooth muscle myosin heavy chain (SmMHC), and calreticulin in the cells were measured on Western blots. RESULTS: Compared with WT IL-13, treatment with the IL-13 R110Q variant resulted in a significant increase in eotaxin release as well as significant, although modest, increases in the expression levels of alpha-SMA, SmMHC, calreticulin, and FcepsilonRI alpha-chain. CONCLUSIONS: The results of the present study suggenst that the IL-13 R110Q variant may enhance enhanced functional activities in hBSMCs.
Actins ; Asthma ; Calreticulin ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin E ; Interleukin-13 ; Interleukins ; Muscle, Smooth ; Myocytes, Smooth Muscle ; Myosin Heavy Chains ; Proteins

OBJECTIVETo observe the effect of amino acid substitution in conserved sequence of penicillin-binding protein (PBP) 1A, 2B, 2X on antimicrobial activity of beta-lactams against Streptococcus pneumoniae (SP).

METHODMinimal inhibitory concentration (MIC) of 6 beta-lactams was determined by the E-test in 59 SP strains. The penicillin-binding protein genes pbp1a, 2b, 2x in every SP strain were amplified by nested-polymerase chain reaction (nPCR), then the PCR products were sequenced using automatic genetic analyzer directly. To analyze the amino acid substitutions, the DNA sequences were converted to protein sequences and aligned by Clustalx software. According to amino acid substitution in conserved sequence of PBP2B, 3 phenotypes were observed, including: PBP2B phenotype I (no amino acid substitution); PBP2B phenotype II (Glutamine 432-->Leucine and/or Threonine 445/451-->Alanine/Serine, Glutamic 481-->Glycine, 1 strain had proline insertion between residues 431/432); PBP2B phenotype III (Alanine 624-->Glycine with the addition of phenotype II). According to amino acid substitution in conserved sequence of PBP1A, 3 phenotypes were observed, including: PBP1A phenotype I (no amino acid substitution); PBP1A phenotype II (Threonine 574-->Asparagine, Serine 575-->Threonine, Glutamine 576-->Glycine, Phenylalanine 577-->Tyrosine, 574TSQF-->NTGY); PBP1A III (Threonine 371-->Alanine/Serine, Proline 432-->Threonine with the addition of 574TSQF-->NTGY). According to amino acid substitution in conserved sequence of PBP2X, 4 phenotypes were observed, including: PBP2X phenotype I (no amino acid substitution); PBP2X phenotype II (Histidine 394-->Leucine or Threonine 338-->Alanine); PBP2X phenotype III (Threonine 338-->Alanine, Isoleucine 371-->Threonine, Arginine 384-->Glycine and Leucine 546-->Valine); PBP2X phenotype IV (Methionine 339-->Phenylalanine, Methionine 400-->Threonine with the addition of PBP2X phenotype III).

RESULTAmong 59 SP strains antibacterial activities distribution (sensitive strains, intermediate strains and resistant strains) of 6 beta-lactams were penicillin (12, 29, 18); amoxicillin(49, 9, 1); cefuroxime (16, 16, 27); ceftriaxone (47, 1, 11); cefotaxime (47, 3, 9); imipenem (49, 10, 0). beta-lactam antibiotics insensitive strains (intermediate + resistant strain) in PBP2B phenotype III, PBP1A phenotype III, PBP2X phenotype III and IV were significantly increased, the MIC(50) of these strains were significantly higher than that of the others.

CONCLUSIONThe amino acid substitutions in or vicinal conserved sequence of PBP of SP increase MIC for beta-lactam antibiotics.

Amino Acid Substitution ; Aminoacyltransferases ; genetics ; Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; genetics ; Microbial Sensitivity Tests ; Penicillin-Binding Proteins ; genetics ; Peptidyl Transferases ; genetics ; Streptococcus pneumoniae ; drug effects ; beta-Lactam Resistance ; genetics ; beta-Lactams ; pharmacology

OBJECTIVETo investigate serum procalcitonin (PCT) concentrations in premature infants with different gestational ages at different times after birth.

METHODSA total of 217 neonates without infection, including 102 premature infants and 115 full-term infants, were enrolled in this study. The premature infants were further divided by gestational age into three subgroups: 30-32 weeks (n=30), 33-34 weeks (n=35) and 35-36 weeks (n=37). All the infants were studied to evaluate serum PCT concentrations at 0-12, 13-24, 25-36, 37-48, 49-72, 73-96, 97-120 and 121-144 hours after birth.

RESULTSIn the newborns, serum PCT concentrations increased gradually after birth, reached peak values at about 24 hours after birth, and then gradually declined and dropped to normal values for children at about 96 hours after birth. In the premature infants, serum PCT concentrations reached peak values at about 36 hours after birth, later than in the full-term infants, then declined slowly and dropped to levels similar to the full-term infants at 96 hours after birth. Serum PCT concentrations in the 30-32 week subgroup remained at low levels after birth, and increased gradually, later than in other premature infants, at 37-48 hours after birth.

CONCLUSIONSEarly after birth, neonates have a changing serum PCT concentration, increasing first and then decreasing. Peak serum PCT levels appear later in premature infants than in full-term infants. Serum PCT concentrations of premature infants with a gestational age of under 32 weeks remain at relatively low levels within 36 hours after birth.

Calcitonin ; blood ; Calcitonin Gene-Related Peptide ; Gestational Age ; Humans ; Infant, Newborn ; Infant, Premature ; blood ; Protein Precursors ; blood ; Time Factors

This study was purposed to establish the BALB/c mouse model with similar diffuse large B cell lymphoma and to investigate the immuno-characteristics of this model. The experiments were divided into 3 groups including group 1 (BALB/c mice with tumor resulting from injection of A20 cells), group 2 (BALB/c mice without tumor formation resulting from injection of A20 cells) and group 3 (normal BALB/c mice). The CD antigen expression of tumor cells in vitro and in vivo, and the ratios of T/B lymphocytes in peripheral blood and spleen tissue of 3 groups were detected by flow cytometry. The results showed that the animal models were successfully established, the pathologic characteristic of tumor cells from animal model were similar to human diffuse large B cell lymphoma. The positive expression levels of CD3, CD4, CD8, CD19 and CD30 in tumor tissue were (49.27 +/- 23.75)%, (6.07 +/- 3.65)%, (51.2 +/- 23.1)%, (67.06 +/- 16.39)% and (37.93 +/- 17.03)% respectively; as compared with A20 cells, the expression levels of CD3 and CD8 significantly increased, while the expression level of CD19 significantly decreased (p < 0.05). The obvious decrease of CD3 and CD4 expression was observed in peripheral blood of mice with tumor as compared with normal mice (p < 0.05). The expression levels of CD3, CD4 and CD8 decreased while the expression level of CD19 increased in the spleen cells of mice without tumor formation as compared with normal mice (p < 0.05). It is concluded that the immunophenotypes of A20 cells and successfully established animal models can be useful studying human B cell lymphoma.
Animals ; B-Lymphocytes ; cytology ; Cell Line, Tumor ; Disease Models, Animal ; Immunophenotyping ; Lymphoma, Large B-Cell, Diffuse ; immunology ; Mice ; Mice, Inbred BALB C ; Mice, Nude