Objective To explore the role of extracellular signal-regulated kinase (ERK)in central nucleus of amygdala (CeA)in the mechanism of fentanyl-induced hyperalgesia (OIH)in rats. Methods Step 1:12 healthy male Sprague-Dawley rats,weighing 60-100 g,were randomly divided into OIH and Control group.The mechanical paw withdrawal threshold (PWT)and the thermal paw withdrawal latency (PWL)were tested at pre-and post-OIH induction.Then the level of p-ERK in the CeA was analyzed by Western blotting.Step 2:After successful induction of OIH and catheterization in CeA,another 30 SD male rats were randomly divided into 5 groups (n = 6 each):Group OIH;Group OIH+U0124;Group OIH+U0126(0.1 5 nmol);Group OIH+U0126(0.45 nmol)and Group OIH+U0126 (1.5 nmol),then 0.3 μl of DMSO,U0124 (1.5 nmol),U0126 (0.1 5 nmol,0.45 nmol,1.5 nmol)was given through the catheter separately.PWT and PWL were tested before cathe-terization,at pre-OIH induction,post-OIH induction and 0.5 h after CeA drug administration.After the last test of pain threshold,the rats were sacrificed and CeA tissues were sampled for analyzing the expression of p-ERK by western blot.Results In step 1 compared with control group,PWT and PWL of OIH group were sharply decreased post-OIH induction (P <0.05),concomitant increase of the expression of p-ERK in CeA in OIH group was also observed.In step 2,both PWT and PWL were sharply decreased post-OIH induction (P <0.05).Intra-CeA U0126 injection,but not U0124, reversed both behavioral hyperalgesia and molecular activation of ERK in CeA in a dose-dependent manner (P <0.05).Conclusion ERK plays a pivotal role in the maintenance of fentanyl-induced hy-peralgesia.Targeting inhibition of ERK activation in CeA can alleviate fentanyl-induced hyperalgesia.

Objective To investigate the infection state and genotype of Babesia from tick at Alataw port,the China-Kazakhstan border.Methods Drag-flag method and animal body surface method were used to collect ticks at Ebinur Lake wetland,Alataw port.18s rRNA gene of Babesia was tested by polymerase chain reaction (PCR),the sequence analysis was conducted with Blast and phylogenetic analysis was conducted with Mega 6.0.Results The positive rate of Babesia gene in ticks was 12.65％ (32/253) at Alataw port.By sequencing,32 sequences were divided in ALSK174,ALSK191,ALSK019 three groups.Analysis of Blast showed that ALSK174 had the highest homology with Babesia caballi (EU888904,South Africa),it was 99.72％ (356/357);ALSK191 had the highest homology with Babesia occultans (KP745626,Turkey),it was 99.72％ (350/351);ALSK019 had 95.76％ (339/354) homology with Babesia odocoilei (KC460321,Canada).Conclusion In this study,we have first reported that the Babesia is infected from ticks at Alataw port,the China-Kazakhstan border.

Abstract: Objective　To investigate a poisoning incident caused by eating eight treasure congee, and establish liquid chromatography (LC)-mass spectrometry (MS)/MS screening method of 28 alkaloids to provide references for disposal of similar poisoning incidents. Methods　LC-MS/MS was used for screening 28 alkaloids in the urine, eight treasure congee and food raw material, and the detected alkaloids were quantified. Samples were extracted with 0.4% formic acid aqueous solution and separated by a Acquity UPLC BEH C18 column (1.7 μm, 100 × 2.1 mm). Acetonitrile-0.2% formic acid aqueous solution was used as the mobile phase and gradient elution was adopted. The ionization mode was electrospray positive ionization mode, and the detection method was multi-reaction monitoring (MRM). Analytes were quantified with the external standard method. Results　In the concentration range of 0-100 ng/mL, the linear correlation coefficient r were greater than 0.999 for 28 alkaloids. The recovery of 28 alkaloids in urine sample ranged from 63.0% to 105.0%, and the relative standard deviations (RSDs) were between 5.8% and 8.6%. The recovery of 28 alkaloids in eight treasure congee sample ranged from 72.0% to 109.0%, and the RSDs were between 6.3% and 9.7%. The recovery of 28 alkaloids in semen sesami nigrum sample ranged from 60.0% to 95.0%, and the RSDs were between 4.8% and 8.2%. Hyoscyamine (2 380.0 ng/mL), scopliamine (3.6 ng/mL) and rac-anisodamine (4.7 ng/mL) were detected in the patient's urine. Hyoscyamine (63.3 μg/g), scopliamine (5.7 μg/g) and rac-anisodamine (2.1 μg/g) were detected in eight treasure congee. Hyoscyamine (901.0 μg/g), scopliamine (80.0 μg/g) and rac-anisodamine (30.1 μg/g) were detected in the seed of Datura stramonium L. The ratio of scopliamine and hyoscyamine in the seed of D. stramonium was 1∶11, which complies with the characteristics of D. stramonium L. In urine sample, the proportion of scopliamine and rac-anisodamine was 0.15% and 0.20%, and hyoscyamine accounted for 99.65%. Conclusion　Seed morphology, the content range and proportion of three alkaloids are all in accord with the characteristics of D. stramonium. Combined with the clinical symptoms of atropine poisoning, it can be deduced that this incident is a family food poisoning caused by accidental consumption of seed of D. stramonium L. The method can provide technical support for the clinical diagnosis and treatment of alkaloid poisoning patients, and also provide a basis for emergency detection and disposal of alkaloid poisoning events.

4-coumarate coenzyme A ligase is a key enzyme of phenylpropanoid metabolic pathway in higher plant and may regulate the biosynthesis of ferulic acid in Angelica sinensis. In this study, the homology-based cloning and rapid amplification of cDNA ends (RACE) technique were used to clone a full length cDNA encoding 4-coumarate coenzyme A ligase gene (4CL), and then qRT-PCR was taken for analyzing 4CL gene expression levels in the root, stem and root tissue at different growth stages of seedlings of A. sinensis. The results showed that a full-length 4CL cDNA (1,815 bp) was obtained (GenBank accession number: KT880508) which shares an open reading frame (ORF) of 1 632 bp, encodes 544 amino acid polypeptides. We found 4CL gene was expressed in all tissues including leaf, stem and root of seedlings of A. sinensis. The expressions in the leave and stem were increased significantly with the growth of seedlings of A. sinensis (P < 0.05), while it in the root showed little change. It indicates a time-space pattern of 4CL gene expression in seedlings of A. sinensis. These findings will be useful for establishing an experiment basis for studying the structure and function of 4CL gene and elucidating mechanism of ferulic acid biosynthesis and space-time regulation in A. sinensis.
Amino Acid Sequence ; Angelica sinensis ; genetics ; Base Sequence ; Cloning, Molecular ; Coenzyme A Ligases ; genetics ; DNA, Complementary ; chemistry ; Molecular Sequence Data

Objective:A new recombinant baculovirus transfer vector was constructed, in whic h a recombinant gene fragment encoding HIV-1 gag-gp120 chimeric gene was inse rted.Methods:After HIV-1 gag gene and gp120 gene were linked,the recombinant baculov irus vector was constructed,and the DNA recombinant technique and the E coli /baculovirus system were used.Results:Gel electrophoresis of DNA analysis showed that the genes were recombine d correctly.Electron microscope showed that the recombinant baculovirus reproduc ed in a great quantity.Conclusion:Recombinant baculovirus vector which HIV-1 gag-gp120 chimeric gene fragment was inserted in was constructed successfully.This vector is useful in study of the expression and the biological function of the HIV-1 Gag-gp120 ch imeric protein.

OBJECTIVEFragile X syndrome (FXS) may be identified by many methods, such as PCR assay and Southern blot. However, each method has its limits or shortcomings. This study explored the reliability of the rapid, convenient and inexpensive hair root fragile X mental retardation protein (FMRP ) assay in the identification of FXS.

METHODSFMRP in hair roots was determined by immunohistochemistry assay in 80 healthy children, in 40 children with mental retardation of unknown etiology and in 12 family members in one pedigree of FXS. FXS was confirmed by 7-deza-dGTP PCR.

RESULTSThere was a high expression of FMRP in hair roots (> or =80%) in healthy children. Two children were confirmed with FXS by 7-deza-dGTP PCR in 40 children with mental retardation of unknown etiology. FMRP expression was 10% and zero respectively in the two children. The other 38 children had FMRP expression of more than 80%. FMRP was not expressed in the two cases of FXS from the pedigree of FXS.

CONCLUSIONSInexpensive, rapid and convenient hair root FMRP assay is reliable for the diagnosis of FXS and may be widely applied for screening and diagnosing FXS in children with mental retardation.

Adolescent ; Child ; Child, Preschool ; Female ; Fragile X Mental Retardation Protein ; analysis ; Fragile X Syndrome ; diagnosis ; genetics ; Hair ; chemistry ; Humans ; Infant ; Male ; Polymerase Chain Reaction

OBJECTIVETo explore the characteristics of B cell-activating factor (BAFF) secreted by peripheral blood monocyte-derived dendritic cell (MoDC) in chronic idiopathic thrombocytopenic purpura (cITP) and the function of MoDC on B cell proliferation.

METHODSTen cITP patients were studied dynamically before and after treatment. The BAFF levels in serum and the supernatant of LPS stimulated MoDC were tested with ELISA. The BAFF gene expression in LPS stimulated MoDC was tested with RQ-PCR, the B cell proliferation co-cultured with the supernatant of LPS stimulated MoDC for 5 days was tested with flow cytometry for CFSE and (3)H thymidine incorporation.

RESULTSThe BAFF level in serum (serum BAFF) \[(2461 ± 483) ng/L\], and supernatant of LPS stimulated MoDC (supernatant BAFF) \[(1113 ± 113) ng/L\] and BAFF mRNA in LPS stimulated MoDC (BAFF mRNA) (1.70 ± 0.23) before treatment were higher than that after treatment \[(621 ± 53) ng/L, (490 ± 49) ng/L and 0.37 ± 0.12\] and normal group \[(742 ± 77) ng/L, (582 ± 63) ng/L and 0.52 ± 0.08\]. There was a positive correlation among serum BAFF, supernatant BAFF and BAFF mRNA, and a negative correlation among serum BAFF, supernatant BAFF and BAFF mRNA and blood platelet count (BPC) in all ITP patients. The supernatant of LPS-stimulated MoDC from untreated patients enhanced B cell proliferation as compared with the supernatant of LPS-stimulated MoDC from treated patients and normal group.

CONCLUSIONBAFF might contribute to disease development in cITP. MoDC may directly increase B cell proliferation by secreting BAFF without T cell help, playing an important role in the antibody production in cITP.

B-Lymphocytes ; immunology ; Dendritic Cells ; immunology ; Humans ; Interleukin-4 ; Monocytes ; Purpura, Thrombocytopenic, Idiopathic ; immunology

OBJECTIVETo observe the effect of Compound Zhajin Granule (CZG) on Toll-like re-ceptor 4 (TLR4) signaling pathway in high-fructose corn syrup induced NASH mice.

METHODSThirty 6-week-old male C3H mice were divided into the high fat and high fructose (HFHFr) group (n = 20) and the control group (n = 10) according to body weight. Mice in the HFHFr group ate high fat diet and drank 20% fructose water, while those in the control group ate common diet and drank common water. After 8 weeks mice in the HFHFr group were divided into two group according to body weight, the HFHFr group and the CZG group, 10 in each group. Mice in the CZG group were fed with high fat forage and 20% fructose water, and administered with 50 mL/kg 12. 8% CZG (prepared by hawthorn, Radix Curcumae, Alisma Orientale, Fritillaria Thunbergii, Silybum Marianum, peach seed in the ratio of 3:1.5:1.5:2:1.5:2:1) by gastrogavage. Mice in the HFHFr group were fed in the same way and daily administered with equal volume of distilled water by gastrogavage. Sixteen weeks later all mice were sacrificed. Body weight, liver wet weight, liver function, and lipid metabolism were detected. Pathological changes of liver tissues were assessed by HE staining, oil red O staining, and Masson staining. Expressions of TLR4, myeloid differentiation factor 88 (MyD88), tumor necrosis factor-alpha (TNF-α) were detected using immunohistochemical staining and real-time fluorescent quantitative PCR.

RESULTSBody weight, alanine aminotransferase (ALT), aspartate aminotransferase (AST) were obviously lower in the CZG group than in the HFHFr group (P < 0.05); oil red O stained area and density were decreased more in the CZG group than in the control group. HE staining showed ballooning inflammation was reduced more in the CZG group than in the HFHFr group. Masson staining was negative. Positive rates of TLR4 and MyD88 and mRNA expressions were significantly lower in the CZG group than in the HFHFr group (all P < 0.05).

CONCLUSIONCZG could significantly inhibit TLR4 signaling pathway of liver in NASH mice.

Alanine Transaminase ; metabolism ; Animals ; Aspartate Aminotransferases ; metabolism ; Diet, High-Fat ; Drugs, Chinese Herbal ; pharmacology ; Fructose ; administration & dosage ; adverse effects ; Inflammation ; Lipid Metabolism ; Male ; Mice ; Mice, Inbred C3H ; Myeloid Differentiation Factor 88 ; metabolism ; Non-alcoholic Fatty Liver Disease ; drug therapy ; Signal Transduction ; drug effects ; Toll-Like Receptor 4 ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVEEstablish the model of mouse with chronic hepatitis B virus (HBV) and nonalcoholic fatty liver disease (NAFLD).

METHODSTake 100 HBV transgenic, BALB/c mice of 4 weeks old, with each gender half. Then pick out 70 mice in one group to feed high-fat feed and the rest to feed normal feed. At the end of week 16, random kill 10 mice of high-fat, then liver tissue and serological detection target identification model is established in this paper. After that, divide the mice into model group and comparison group with 30 mice in each group. Feed model group with high-fat feed, comparison group with normal feed and normal group with normal feed till week 72 (including previous 16 weeks). Kill 10 mice of each group at the end of week 24, 48 and 72 respectively, fully automatic biochemical instrument detection of serum ALT, AST, TC, TG, FBG, fluorescence quantitative PCR method to detect HBV-DNA, chemiluminescence detection of HBsAg, liver biopsy after HE staining to evaluate histology change, observe mice model of dynamic evolution.

RESULTS(1) Feed high fat feed after 16 weeks, mice's weight, serum ALT, AST, TC, TG, FBG and blood biochemical indicators increased, HBV-DNA positive, liver HE staining obviously big blister fatty degeneration of liver cells and within the lobule lymphocytes infiltration, NAFLD activity score (NAS) getting close to NASH, the model of chronic HBV carries with NAFLD mouse built successfully. (2) The TC and TG values of model group in each period were higher than that of comparison group and normal group. (3) In week 24 and 72, HBV-DNA values of each group are obvious different from the other two groups and the difference can be applied to statistical significance (P < 0.05). (4) In week 48 and 72, NAS of each group are obvious different from the other two groups and the difference can be applied to statistical significance (P < 0.05).

CONCLUSIONS(1) Chronic HBV carries with NAFLD mice model can be established by HBV transgenic mice fed by high fat feed. (2) NAFLD accelerates the liver disease of the mice carrying HBV to some extent.

Animals ; Disease Models, Animal ; Fatty Liver ; complications ; pathology ; virology ; Female ; Hepatitis B virus ; genetics ; isolation & purification ; physiology ; Hepatitis B, Chronic ; complications ; pathology ; virology ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Transgenic ; Non-alcoholic Fatty Liver Disease

OBJECTIVETo study the differential expression of four TRAIL receptors on bone marrow mononuclear cells (BMMNC) from multiple myeloma (MM) patients and myeloma cell line KM3 cells, to compare their altered expressions after chemotherapy and to explore the mechanisms by which TRAIL selectively kills tumor cells.

METHODSSemi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry were used to investigate the expression of four TRAIL receptors on BMMNCs in 23 MM patients, KM3 cells and 15 controls, and the changes of their expression pattern after chemotherapy and after incubation of KM3 cells with sub-clinical concentration of doxorubicin.

RESULTSDR4 and DR5 were highly expressed on KM3 cells with no expression of DcR1 and DcR2. Expressions of DR4 and DR5 on BMMNCs from MM patients were higher and expression of DcR1 and DcR2 were lower than that of controls (P < 0.05). The expression of DR5 on MM and KM3 cells was up-regulated after chemotherapy and exposure to doxorubicin (P < 0. 05).

CONCLUSIONSThe expressions of four TRAIL receptors on myeloma cells and normal controls were different, which might account for the selective killing effect of TRAIL on MM cells. Up-regulated DR5 on KM3 cells after incubating with doxorubicin and after chemotherapy suggests the cytotoxic agents might enhance the apoptosis of MM cells.

Antineoplastic Agents ; pharmacology ; Cell Line, Tumor ; Cells, Cultured ; Doxorubicin ; pharmacology ; Female ; Flow Cytometry ; Gene Expression ; drug effects ; Humans ; Leukocytes, Mononuclear ; cytology ; drug effects ; metabolism ; Male ; Middle Aged ; Multiple Myeloma ; drug therapy ; genetics ; pathology ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction