Adolescent ; Adult ; Aged ; Aged, 80 and over ; Carcinoma, Hepatocellular ; diagnosis ; epidemiology ; virology ; Child ; China ; epidemiology ; Female ; Genes, Viral ; Genotype ; Hepatitis B virus ; genetics ; Humans ; Liver Neoplasms ; diagnosis ; epidemiology ; virology ; Male ; Middle Aged ; Viral Load ; Young Adult

The aim was to construct a prokaryotic expression plasmid encoding the fusion gene of single-chain variable fragment and monocyte chemotactic protein-3 (MCP-3). The cDNAs of immunoglobulin (Ig) VH and Ig VL were amplified by RT-PCR and assembled into the single-chain variable fragment (scFv) by recombinant PCR method. The cDNAs of Ig VH and Ig VL were connected by a (Gly4Ser)3 linker. Then, the fragments of scFv and MCP-3 were connected with a NDAQAPKS spacer, using recombinant PCR method again. The results indicated that the fusion gene of scFv-MCP-3 were constructed correctly and cloned into the prokaryotic expression plasmid successfully identified by sequencing and restriction endonucleases examination. Finally, the fusion protein was expressed in E coli DH5alpha under induction by arabinose. And the fusion protein was 65 kD and account for 30% of the total protein of the bacteria. In conclusion, a prokaryotic plasmid, encoding the fusion gene of single-chain variable fragment with MCP-3 and expressing idiotype protein vaccination against B cell lymphoma, was constructed correctly.
Amino Acid Sequence ; Animals ; Cancer Vaccines ; biosynthesis ; genetics ; immunology ; Chemokine CCL7 ; biosynthesis ; genetics ; immunology ; Genetic Vectors ; Humans ; Immunoglobulin Idiotypes ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Lymphoma, B-Cell ; immunology ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Plasmids ; genetics ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Vaccines, DNA ; biosynthesis ; genetics ; immunology

OBJECTIVETo investigate the mechanism of ethanol-induced calcium overload in hepatocytes and the related role of store-operated calcium channels (SOCs).

METHODSHepG2 cells were treated an ethanol concentration gradient with or without intervention treatment with the extracellular calcium chelator EGTA or the SOCs inhibitor 2-aminoethoxydiphenyl borate (2-APB). Effects on cell viability were assessed by the CCK8 assay. Effects on leakage of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined by automatic biochemical analyzer measurements of the culture supernatants. Effects on cytoplasmic free Ca2+ concentration ([Ca2+]i) were accessed by detecting fluorescence intensity of the calcium indicator Fluo-3/AM with a flow cytometer. Effects on mRNA and protein expression levels of SOCs, stromal interacting factor 1 (STIM1), and calcium release-activated calcium channel protein 1 (Orai1) were evaluated by qPCR and western blotting.

RESULTSThe ethanol treatment produced dose-dependent reduction in cell viability (r = -0.985, P less than 0.01) and increases in leakage of ALT (F = 15.286, P less than 0.01) and AST (F = 39.674, P less than 0.01). Compared to untreated controls, the ethanol treatments of 25, 50, 100, 200 and 400 mM induced significant increases in [Ca2+]i level (1.25+/-0.36, 1.31+/-0.15, 1.41+/-0.18, 2.29+/-0.25, 2.58+/-0.19; F = 15.286, P less than 0.01). Both intervention treatments, EGTA and 2-APB, significantly reduced the 200 mM ethanol treatment-induced [Ca2+]i increase (2.32+/-0.08 reduced to 1.79+/-0.15 (t = 7.201, P less than 0.01) and 1.86+/-0.09 (t = 8.183, P less than 0.01) respectively). EGTA and 2-APB also increased the ethanol-treated cells' viability and reduced the ALT and AST leakage. The 200 mM ethanol treatment stimulated both gene and protein expression of STIM1 and Orai1, and the up-regulation effect lasted at least 72 h after treatment.

CONCLUSIONEthanol-induced dysregulation of SOCs may be an important molecular mechanism of ethanol-induced [Ca2+]i rise in hepatocytes and the related liver cell injury.

Calcium ; metabolism ; Calcium Channels ; metabolism ; Ethanol ; adverse effects ; Hep G2 Cells ; Hepatocytes ; drug effects ; metabolism ; Humans

OBJECTIVETo investigate the features of white matter myelin development in preterm infants using magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI).

METHODSA total of 31 preterm infants with a gestational age of ≤32 weeks and a birth weight of <1 500 g were enrolled. According to head MRI findings, these infants were divided into preterm group with brain injury (12 infants) and preterm group without brain injury (19 infants). A total of 24 full-term infants were enrolled as control group. Head MRI and DTI were performed at a gestational age or corrected gestational age of 37-40 weeks. Fractional anisotropy (FA) and apparent diffusion coefficient (ADC) were measured for the same regions of interest in the three groups.

RESULTSThe preterm group with brain injury showed a significantly lower FA value of the posterior limb of the internal capsule than the preterm group without brain injury and full-term control group (P<0.05). The preterm groups with and without brain injury showed significantly lower FA values of frontal white matter and lenticular nucleus than the full-term control group (P<0.05). The FA value of occipital white matter showed no significant differences among the three groups (P>0.05). Compared with the full-term control group, the preterm groups with and without brain injury showed significantly higher ADC values of the posterior limb of the internal capsule, lenticular nucleus, occipital white matter, and frontal white matter (P<0.05).

CONCLUSIONSAfter brain injury, preterm infants tend to develop disorder or delay of white matter myelination in the posterior limb of the internal capsule. At a corrected full-term gestational age, the preterm infants with and without brain injury have a lower grade of maturity in periventricular white matter and grey matter than full-term infants.

Brain Injuries ; physiopathology ; Diffusion Tensor Imaging ; methods ; Humans ; Infant, Newborn ; Infant, Premature ; physiology ; Magnetic Resonance Imaging ; methods ; Myelin Sheath ; physiology ; White Matter ; growth & development

OBJECTIVETo study the relationship between rs6267 polymorphism of catechol-O-methyltransferase (COMT) gene and attention deficit hyperactivity disorder (ADHD).

METHODSOne hundred and fourteen children with ADHD and 76 normal volunteers were enrolled. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques were used for detecting COMT rs6267 polymorphism. The behavioral problems were assessed by Child Behavior Checklist (CBCL).

RESULTSThere were no significant differences in the COMT genotype distribution and allele frequencies between the ADHD and normal control groups. The frequencies of thinking problems (1.7±1.9 vs 1.0±0.9) and disciplinary problems (4.5±3.7 vs 2.2±1.4) in ADHD children carrying genotype G/G were significantly higher than those in children carrying G/T (P<0.05).

CONCLUSIONSCOMT rs6267 polymorphism may not contribute to susceptibility to ADHD. However, there might be an association between rs6267 polymorphism and some clinical characters of ADHD.

Adolescent ; Attention Deficit Disorder with Hyperactivity ; genetics ; Catechol O-Methyltransferase ; genetics ; Child ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Male ; Polymorphism, Genetic

Objective To explore the effect of dendritic cells (DC) modified with transforming growth factor β1 (TGF-β1) gene on the expressions of CD28/CTLA-4:B7 costimulatory molecules in peripheral blood mononuclear cells (PBMC) in the Lewis rats with experimental autoimmune myasthenia gravis (EAMG). Methods Thirty inbreeding line, healthy, female Lewis rats were divided randomly into 6 groups: normal group, EAMG group, DC treatment group, pcDNA3-TGF-β1-DCtreatment group, pcDNA3-DC control group and normal saline group. The rats were immunized with the AChR protein extracted from electric organ of Narcine timilei and CFA in the groups except normal group. 2×106 pcDNA3-TGF-β1-DCs/rat were injected subcutaneously into the backs of the rats which had been immunized 5 d earlier with AChR+CFA. The rats in DC treatment group, pcDNA3-DC control group and normal saline group were injected in parallel with untreated DC, pcDNA3-DC and normal saline, respectively. Seven weeks after the first immunization, the expressions of CD28 mRNA and CTLA-4 mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR), and the levels of B7-1 and B7-2 on the surface of PBMC were examined using flow cytometry. Results (1)The low expression of CD28 mRNA and rare expression of CTLA-4 mRNA were found in the normal rats, and both expressions increased markedly in EAMG rats (P＜0.001). Compared to those in EAMG group, the expression of CD28 mRNA decreased and CTLA-4 mRNA was upregulated after the treatment with pcDNA3-TGF-β1-DC (P＜0.05). There was no significant difference in the expressions of CD28 mRNA and CTLA-4 mRNA among the EAMG group, DC treatment group, pcDNA3-DC control group and normal saline group (P＞0.05). (2) The expressions of CD28, CTLA-4, B7-1 and B7-2 on the surface of PBMC were rare in normal rats, which increased significantly in EAMG rats (P＜0.001). The levels of CD28, B7-1 and B7-2 in pcDNA3-TGF-β1-DC group were lower than those in EAMG group (P＜0.01), but the level of CTLA-4 was higher than that in EAMG group (P＜0.05). They showed no statistically difference among the EAMG group, DC treatment group, pcDNA3-DC control group and normal saline group (P＞0.05). Conclusions The expressions of CD28/CTLA-4:B7 costimulatory molecules are abnormal in the rats with EAMG. The regulation of CD28/CTLA-4:B7 costimulatory pathways may play a critical role in the mechanism of the treatment with DC transfected with pcDNA3-TGF-β1 in the incipient EAMG rats.

Objective To investigate the effect of agonist CD40 monoclonal antibody (CD40mAb) on the tolerance of experimental autoimmune myasthenia gravis (EAMG) induced by immature dendritic cells (iDCs). Methods Lewis rats were equally randomized into normal group,EAMG group, tolerance group and CD40mAb treatment group (n=5). Rats in the tolerance group and CD40mAb treatment group were vaccinated with AChR pulsed iDCs; and rats in the CD40mAb treatment group were intraperitoneally injected CD40mAb at a dosage of 0.5 mg once when performing the vaccination and on the 2rd d of vaccination. One mL serum-free medium was given to the rats in the EAMG group; normal group did not receive any treatment. Three weeks after that, rats in the above 4 groups were immunized with AChR and complete Freund's adjuvant (CFA). Seven weeks after the immunization, the corresponding indexes of MG were observed: behavioral assessment was performed and electromyogram was employed to detect the repetitive nerve stimulation on these rats; enzyme-linked immunosorbent assay (ELISA) was used to determine the level of AChRab; the pathological changes of neuromuscular junction were also detected. Results Just as the rats in the normal group, the rats in the tolerance group did not have significant changes in any of the corresponding indexes of MG after being immunized with AChR and CFA. In contrast, rats in both EAMG group and CD40mAb treatment group showed typical changes in the corresponding indexes of MG: their electromyogram wave amplitude obviously attenuated; the level of serum AChRab increased and neuromuscular junction appeared as a typical damage of MG. Conclusion Agonist CD40mAb could abrogate the tolerance of EAMG induced by AChR pulsed iDCs, suggesting that the dysfunction of DCs is related to the priming of abnormal immune of MG.

Objective To study the family rearing pattern of attention deficit hyperactivity disorder(ADHD)with or without anxiety disorder and to explore its risk factors.Methods 9495 children and their parents were sampled at random in Hunan province,using two-stage investigation.Those who were diagnosed ADHD and the normal control filled out Egna Minnen av Barndoms Uppfostran and family adaptability and cohesion scale bv themselves.Results The comparison of factors as:actual family cohesion,parents' punishments,reiection,mother's excessive protection,intervention and father's excessive protection were significantly difierent between ADHD with or without anxiety disorder and normal children(P＜0.05).The comparison of parents' punishments,reiection,excessive protection and intervention were obviously different between ADHD with anxiety disorder and simple ADHD(P＜0.05).Mother's reiection was the influencing factor of simple ADHD,with OR as 1.122.Ideal family cohesion,mother's rejection and father's punishments were the influencing factors of ADHD with anxiety disorder,with OR as 0.966.1.215 and 1.089 respectively.Conclusion There were some problems in the parental rearing pattern of ADHD with or without anxiety disorder.Mother's rejection,father's punishments and ideal family cohesion were suggested to be correlated with ADHD and anxiety disorder.

OBJECTIVESTo investigate the effects of newborn bull serum(NBS), vitamin C and vitamin E on cryopreservation of mouse seminiferous epithelial cells.

METHODSThe seminiferous epithelial cells from 7-day-old mice were cryopreserved in different freezing solutions. The cell recoveries were examined by Trypan blue exclusive staining after thawing. The freezing solutions composed of DMEM, 10% dimethylsulphoxide(DMSO), and 0, 5%, 10%, or 20% NBS, respectively, or composed of DMEM, 10% DMSO, 10% NBS, and 150 micrograms/ml vitamin C or 50 micrograms/ml vitamin E, respectively.

RESULTSThe cell recoveries in freezing solution containing 0, 5%, 10%, or 20% NBS were 83.4%, 84.7%, 85.7% and 83.6%, respectively. There were no significant differences between them. The cell recoveries in freezing solution containing vitamin C or vitamin E were 88.0% and 82.9%, respectively. There was no significant differences compared with that in freezing solution containing 10% DMSO and 10% NBS.

CONCLUSIONSNBS, vitamin C and vitamin E have no significant protecting effects on mouse seminiferous epithelial cells, and can not significantly improve the cell recoveries.

Animals ; Ascorbic Acid ; pharmacology ; Cattle ; Cryopreservation ; Epithelial Cells ; physiology ; Fetal Blood ; physiology ; Male ; Mice ; Seminiferous Epithelium ; cytology ; Vitamin K ; pharmacology

OBJECTIVETo study the roles of different truncated hepatitis C virus (HCV) core proteins (CORE) in the pathogenesis of HCV persistent infection and hepatocellular carcinoma (HCC) and to assess intracellular localization in transiently transfected cells.

METHODSSeven truncated GFP (green fluorescent protein)-CORE fusion protein expression plasmids were constructed, which contained HCV CORE sequences derived from tumor tissues (BT) and non-tumor tissues (BNT) from one patient infected with HCV. Amino acid (aa) lengths were BT: 1-172 aa, 1-126 aa, 1-58 aa, 59-126 aa, 127-172 aa; BNT: 1-172 aa and C191: 1-172 aa respectively. Subcellular localization of CORE-GFP was analyzed by con-focal laser scanning microscope. Apoptosis and necrosis were quantified by flow cytometry.

RESULTSDifferent truncated CORE-GFP localized mainly in the cytoplasm, but nuclear staining was also observed. HCV CORE could induce apoptosis and necrosis, and different truncated COREs could induce cell apoptosis and necrosis at different levels. Among the same length 1-172 aa of BT, BNT and C191, the cell apoptosis and necrosis percentage of BT is highest, and C191 is the lowest (BT>BNT>C191). To the different fragment COREs of BT, N-terminal of CORE induced apoptosis and necrosis higher, compared with that of C-terminal (1-172 aa>1-126 aa>1-58 aa>127-172 aa>59-126 aa).

CONCLUSIONThese results suggest HCV CORE could induce apoptosis and necrosis of cells, which might play an important role in the pathogenesis of HCV persistent infection and HCC and the different CORE domains of different HCV quasi-species might have some difference in their pathogenesis.

Apoptosis ; Cell Line, Tumor ; Hepacivirus ; genetics ; pathogenicity ; physiology ; Humans ; Necrosis ; virology ; Sequence Deletion ; genetics ; Viral Core Proteins ; chemistry ; genetics ; metabolism