To explore the expression potential of heterogeneous genes using the backbone of infectious bronchitis virus (IBV) Beaudette strain, the ectodomain region of the Spike gene (1,302 bp) of IBV H120 strain was amplified by RT-PCR and replaced into the corresponding location of the IBV Beaudette strain full-length cDNA. This recombinant was designated as BeauR-H120(S1). BeauR-H120(S1) was directly used as the DNA template for the transcription of viral genomic RNA in vitro. Then, the transcription product was transfected into Vero cells by electroporation. At 48 h post-transfection, the transfected Vero cells were harvested, and passaging continued. A syncytium was not observed until the recombinant virus had passed through four passages. The presence of rBeau-H120(S1) was verified by the detection of the replaced ectodomain region of the H120 Spike gene using RT-PCR. Western blot analysis of rBeau-H120 (S1)-infected Vero cell lysates demonstrated that the nucleocapsid (N) protein was expressed, which implied that rBeau-H120(S1) could propagate in Vero cells. The TCIDs0 and EIDs0 data demonstrated that the titer levels of rBeau-H120(S1) reached 10(590+/-0.22)TCID50/mL and 10(6.13+/-0.23)EID50/mL in Vero cells and 9-day-old SPF chicken embryos, respectively. Protection studies showed that the percentage of antibody-positive chickens, which were vaccinated with rBeau-H120(S1) at 7 days after hatching, rose to 90% at 21 days post-inoculation. Inoculation provided an 85% rate of immune protection against a challenge of the virulent IBV M41 strain (103EID50/chicken). This recombinant virus constructed using reverse genetic techniques could be further developed as a novel genetic engineering vaccine against infectious bronchitis.
Animals ; Cercopithecus aethiops ; Chick Embryo ; Chickens ; Coronavirus Infections ; veterinary ; virology ; Infectious bronchitis virus ; chemistry ; genetics ; growth & development ; metabolism ; Poultry Diseases ; virology ; Protein Structure, Tertiary ; Spike Glycoprotein, Coronavirus ; chemistry ; genetics ; metabolism ; Transfection ; Vero Cells

Child, Preschool ; Chondromatosis, Synovial ; diagnosis ; diagnostic imaging ; surgery ; Diagnostic Errors ; Female ; Humans ; Tibial Fractures ; diagnosis ; Tomography, X-Ray Computed

Carcinoma, Hepatocellular ; surgery ; Catheter Ablation ; methods ; Humans ; Liver Neoplasms ; surgery ; Ultrasonics

Animals ; Astrocytes ; immunology ; metabolism ; Male ; Microglia ; immunology ; metabolism ; Neurons ; metabolism ; Oxidopamine ; metabolism ; Parkinson Disease ; immunology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo evaluate the optimal timing of hepatectomy for intrahepatic lithiasis complicated with acute cholangitis.

METHODSOne hundred and twenty-six patients with hepatolithiasis who had a history of acute cholangitis and underwent hepatectomy were reviewed retrospectively. According to the period between the surgery and last attack of acute cholangitis, 126 patients were divided into 3 groups: > 3 months (group A, n = 73), 1 approximately 3 months (group B, n = 28), < 1 month (group C, n = 25). The operation time, blood loss, hospital stay, postoperative complications and stone residual rate were compared among the groups.

RESULTSThe intraoperative blood loss of C group was (644.0 +/- 625.7) ml, which was significantly higher than those of A and B group [(409.2 +/- 250.7) ml and (423.2 +/- 237.1) ml, respectively]. The numbers of patients who needed transfusion and the amount of blood transfusion in group C were also higher than those of group A and B. The incidence rate of complications, residual stone in group C were all markedly higher than those of group A and B. The period of hospital stay in group C was much longer than that in group A and B.

CONCLUSIONSThe optimal timing of hepatectomy for hepatolithiasis complicated with acute cholangitis is at least one month after subsidence of cholangitis.

Adult ; Aged ; Bile Ducts, Intrahepatic ; Cholangitis ; complications ; Cholelithiasis ; complications ; surgery ; Female ; Hepatectomy ; methods ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Time Factors

OBJECTIVETo investigate the effect of DADLE, a δ-opioid receptor agonist, on the proliferation of human liver cancer HepG2 cells and explore the mechanism involving PKC pathway.

METHODSHepG2 cells were treated with DADLE at different doses (0.01, 0.1, 1.0 and 10 µmol/L). Cell viability was determined using methyl thiazolyl terazolium (MTT) assay. The expression of PKC mRNA and p-PKC protein were examined by RT-PCR and Western blot assay. After treated separately with DADLE plusing NAL or PMA, the cell cycle of HepG2 cells was analyzed by flow cytometer. MTT was used to detect their proliferation capacity and Western blot was used to examine the p-PKC expression. The growth inhibitory rate of HepG2 cells treated with DADLE and cis-diammine dichloridoplatinum (CDDP) was analyzed.

RESULTSDADLE at different concentrations showed an inhibitory effect on the proliferation of HepG2 cells though inhibiting the expression of PKC mRNA and p-PKC protein. The results of flow cytometry showed that compared with the control group, the percentage of S + G(2)/M cells in DADLE-treated group was lowered by 3.94% (P < 0.01). Meanwhile, after treated with NAL and PMA, the percentage was elevated by 3.22% and 3.63%, respectively (P < 0.01). The MTT and Western blot assays showed that compared with the control group, the values of A570 and p-PKC protein levels in the HepG2 cells of DADLE-treated group were significantly decreased (P < 0.01). After treatment with NAL and PMA, the values of A570 and p-PKC protein levels were elevated significantly (P < 0.01). The growth inhibitory rate of DADLE + CDDP group was 79.9%, significantly lower than 25.2% and 43.2% of the DADLE and CDDP groups, respectively.

CONCLUSIONSActivation of δ-opioid receptor by DADLE inhibits the apoptosis of human liver cancer HepG2 cells. The underlying mechanism may be correlated with PKC pathway. DADLE can enhance the chemosensitivity of HepG2 cells to CDDP.

Antineoplastic Agents ; pharmacology ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; Dose-Response Relationship, Drug ; Drug Resistance, Neoplasm ; Enkephalin, Leucine-2-Alanine ; administration & dosage ; pharmacology ; Hep G2 Cells ; Humans ; Naltrexone ; analogs & derivatives ; pharmacology ; Phosphorylation ; Protein Kinase C ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Receptors, Opioid, delta ; agonists ; Signal Transduction ; Tetradecanoylphorbol Acetate ; analogs & derivatives ; pharmacology

OBJECTIVETo report a case of pure erythroid leukemia.

METHODSThe clinical features, treatment and prognosis of a rare case of pure erythroid leukemia were reported, and the related literature was reviewed.

RESULTSThe pure erythroid leukemia patient was diagnosed by 90.4% pronormoblasts in bone marrow, 99.5% for erythroid antigen CD71, 67.4% for glycophorin A were detected, while no differentiation antigen of myeloid, lymphoid and megakaryocyte lineages were observed. HAG (homoharringtonine + Cytarabine and G-CSF) regimen were administered with no effect. The patient developed multiple organ failure and died soon.

CONCLUSIONPure erythroid leukemia has a fulminant clinical course with poor response to chemotherapy and worse prognosis.

Humans ; Leukemia, Erythroblastic, Acute ; therapy ; Male ; Middle Aged ; Prognosis

AIMTo further explore the roles of endogenous nitric oxide (NO) or NO derivatives in complex partial seizures and generalized convulsions.

METHODSThe effect of pretreatment with L-nitroarginine (L-NNA), an inhibitor of nitric oxide synthase (NOS), or L arginine (L-Arg), a precursor of NO on kainic acid (KA)-induced seizure in rats and the changes in the concentration of NO2 -/NO- in the hippocampus were determined.

RESULTSThe rats appeared with wet dog shakes (WDS) at 15 min and then occurred generalized convulsions during 1 h to 3 h after administration of KA (10 mg/kg i.p.). However, the pretreatment of L-NNA (50 mg/kg) so dramatically promoted and enhanced KA-induced behavioral seizures that the latency of generalized convulsion was shorten dramatically, and the mortality was greatly high. In contrast, the pretreatment with L-Arg (40 mg/kg) markedly delayed or weakened KA-induced behavioral changes, such as increasing latency of WDS and generalized convulsion, shortening time o f seizure and none of animal died during observed time. The concentration of NO2- /NO3- in the hippocampus increased immediately at 30 min and remained to 7 d after the administration of KA. Compared with control group (pretreatment with NS), the concentration of NO2- / NO3- in the hippocampus apparently increased at 3 h and 3 d after the administration of KA in the rats with L-Arg pretreatment.

CONCLUSIONThe endogenous NO (NO or NO derivatives) mediators may play an important role against excitotoxin induced seizures in rats.

Animals ; Arginine ; pharmacology ; Kainic Acid ; adverse effects ; Male ; Nitric Oxide ; metabolism ; Nitroarginine ; pharmacology ; Rats ; Rats, Wistar ; Seizures ; chemically induced ; metabolism

OBJECTIVEObserving the time course and establishing the model of kappaB-decoy oligodeoxynucleotides (rcB-decoy) inhibiting the activity of NF-kappaB in the PC12 cells.

METHODSPC12 cells cultivating in the 6 wells plate were divided into 3 groups, experimental group: adding kappaB-decoy complex (6 microg DNA/well), the control group: adding scrambled-decoy complex, the normal group: adding lipid-Lipofectamine 2000, transfer and cultivate 48 h, then lipopolysaccharide (LPS, 200 ng/ml) was added in the cells for 0.5-4 h. The immunocytochemistry and Western blot were used to measure the expression or the activity of NF-kappaB in PC12 cells.

RESULTSIn PC12 cells, compared with normal group, the expression of NF-kappaB enhanced obviously with the time of the stimulation of LPS in scrambled-decoy treated control group (P < 0.01), in 2-4 h the level reached the peak; the expression of NF-kappaB showed the stable level with the time of the stimulation of LPS in kappaB-decoy treated experimental group, compared with the control group, the expression levels were obviously lower than the respective time point of control groups (P < 0.01).

CONCLUSIONkappaB-decoy could reduce the expression of NF-kappaB in the normal PC12 cells and inhibit the activity of NF-cB in the pathologic PC12 cells.

Animals ; Cells, Cultured ; Lipopolysaccharides ; pharmacology ; NF-kappa B ; antagonists & inhibitors ; metabolism ; Oligodeoxyribonucleotides ; pharmacology ; PC12 Cells ; Rats

OBJECTIVETo investigate the effect of Suanzao nacute hepatic failure in mice.

METHODAcute liver failure was induced in male Kunming strain mice by enterocoelia injecting the animals with D-Gal-N and LPS. The mice in treatment groups were given corresponding drug 2 h before administration of D-Ga1-N and LPS, and the mice in control group were given the same dose of distilled water. The 24 h survival rate, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels were compared. Serum the levels of TNF-alpha and IL-1 and the levels of SOD, MDA, GR, GSH, NO and NOS in the liver were determined.

RESULTTreatment with suanzaoren decoction could increase the survival rate and improve the liver histological feather. Suanzaoren decoction inhibited the serum the levels of ALT, AST, TNF-alpha and IL-1, and reduced the levels of MDA, NO and NOS and increased the levels of GR and SOD in the liver.

CONCLUSIONTreatment with Suanzaoren decoction can suppress the D-Gal-N/LPS-induced acute hepatic failure. It may be the mechanism that Suanzaoren decocotion regulate the production of inflammatory cytokines and free radicals.

Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Cytokines ; metabolism ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Free Radicals ; metabolism ; Galactosamine ; Glutathione ; metabolism ; Lipopolysaccharides ; Liver ; drug effects ; metabolism ; pathology ; Liver Failure, Acute ; blood ; chemically induced ; prevention & control ; Male ; Malondialdehyde ; metabolism ; Mice ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; Random Allocation ; Superoxide Dismutase ; metabolism