ObjectiveTo observe the behavioral and pain threshold alterations， as well as the changes in indexes related to depression and pain in the serum and central system in mice stressed by maternal separation and chronic neuropathic pain， and explore the underlying mechanism of Wenyang prescription （WY）， Jieyu prescription （JY）， and Wenyang Jieyu prescription （WYJY） in improving depression and pain sensitivity. MethodThe birth date of mice was recorded as PD0. After birth， the mice were divided into a blank group and an experimental group. The neonatal mice in the experimental group underwent maternal separation in PD5-14 at 8 h·d^-1. After ablactation， the mice were divided into a maternal separation group， a WY group （Erxian decoction， 5.84 g·kg^-1）， a JY group （Xiaoyaosan， 12.00 g·kg^-1）， a WYJY group （16.68 g·kg^-1）， and a fluoxetine group （2.60 mg·kg^-1）， with 15 mice in each group. Meanwhile， 15 male mice of the same age without maternal separation were assigned to the normal control group. Mice in the blank group and the maternal separation group were fed on a regular chow diet in PD21-PD90， while the remaining groups were fed on the corresponding drugs. In PD91， sciatic nerve ligation was performed to induce a model of maternal separation and chronic neuropathic pain. The open field test was used to observe the depression-like behaviors of mice in each group， and the mechanical and temperature pain thresholds were measured to detect the pain sensitivity of mice in each group. The serum levels of corticosterone （CORT）， substance P， and β-endorphin （β-EP） were determined by enzyme-linked immunosorbent assay （ELISA）， and the expression of the glucocorticoid receptor （GR） in the amygdala and β-EP protein in the hypothalamus was detected by immunohistochemistry. The mRNA expression levels of amygdala GR gene （Nr3c1）， FK506 binding protein 5 gene （FKBP5）， metabolic glutamate receptor 5 gene （GRM5）， and brain-derived neurotrophic factor （BDNF） were detected by real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）. ResultCompared with the blank group， the maternal separation group showed reduced stay time and total distance traveled in the 5-min open field test （P<0.01）， reduced mechanical pain threshold （P<0.01）， increased serum CORT and β-EP （P<0.01）， declining FKBP5 mRNA expression （P<0.01）， and increased hypothalamic β-EP expression （P<0.05）. Compared with the maternal separation group， the groups with drug intervention showed prolonged stay time （P<0.05， P<0.01） and up-regulated pain thresholds to different degrees. The total distance traveled in the 5-min open field test increased in the WY group， the WYJY group， and the fluoxetine group （P<0.05， P<0.01）. The JY group showed decreased serum CORT （P<0.01）， reduced β-EP ， and increased BDNF mRNA （P<0.01）. Nr3c1 and GRM5 mRNA decreased in the WY group （P<0.05， P<0.01）. The WYJY group showed decreased serum CORT （P<0.05）and decreased Nr3c1， GRM5， and BDNF mRNA （P<0.05， P<0.01）. The levels of β-EP expression were elevated to different degrees in the groups with drug intervention， but the differences were not significant. The levels of GR expression in the WY group， the JY group， and the WYJY group increased （P<0.05）. ConclusionWYJY can inhibit central pain sensitization and regulate hypothalamic-pituitary-adrenal gland （HPA） axis function by enhancing the expression of GR in the amygdala and inhibiting neuroplasticity and excitability in the amygdala to relieve depression-like behaviors and improve somatic hyperalgesia.

Objective To observe the biological activities of human doppel (Dpl) protein transiently expressed and Dpl-like protein PrPΔ32-121 on a human neuroblastoma cell line SH-SY5Y. Methods Recombinant mammalian expression plasmids containing human PRND gene and truncated PrPΔ32-121 fragment were generated by PCR. The expression and location of Dpl and PrPΔ32-121 post-transfection were observed by IFA. The cytotoxicity was measured by MTT analysis. Cellular apoptosis was investigated by flow cytometry and Western blot. Results Both Dpl and PrPΔ32-121 protein were expressed and mainly located on the cell membrane. Remarkable cytotoxicity was detected on SH-SY5Y cells after 24 h transfection. Meanwhile, more Annexin V/PI positively-stained cells as well as lower levels of cellular pro-caspase-3 and Bel-2 were detected in the cells receiving Dpl and PrPΔ32-121 expressing plasmids. Conclusion Dpl protein transiently expressed and PrPΔ32-121 can lead to the similar neural cytotoxicity, probably triggering the cell apoptosis program.

Objective To investigate the cell-type-specific enhancer (CTSE) in HPV16 and its variation in cervical carcinoma. Methods CTSEs were detected by polymerase chain reaction (PCR) in 58 cervical carcinoma from Shaanxi province; in addition variation of CTSEs was analyzed through single-strand conformation polymorphisms (SSCP). Results HPV16 CTSEs were detectable in 34 of 58 (57%) specimens and mutant rate was 41%(14/34) and the main mutations of chosen randomly variant CTSE (CTSEv) happened at YY1 binding sites in addition to glucocoticoid response elements (GRE). Conclusion CTSE in some specimens of Shaanxi province was obviously different from that in HPV16 wild type and variant CTSE might affect the transcriptional regulation of LCR on viral P97, which regulates over-expression of viral oncogenes in cervical carcinoma.

Objective To investigate the relationship between erythrocyte immune function and selenium (Se)level. Methods Forty-nine Kashin-Beck patients in endemic area aged 13- 16 years were divided into two groups and were orally given either selenized yeast or sodium selenite to provide 200 μg selenium per day for 12 weeks. Erythrocyte selenium level, glutathione peroxidase activity, the rosette formation rates of red blood cells complement receptor type Ⅰ(CR1), the immune function of red blood cells, and circulating immune complexes(CIC) were determined. Results After supplementing with selenium for 12 weeks, erythrocyte selenium level, glutathione peroxidase activity, the rosette formation rates of red blood cells CR1 were significantly increased. But the difference in rosette formation rates of IC and CIC content was not significant between before and after Se supplementation. Conclusion The increase of the immune function of the erythrocyte by selenium-supplement may be one of the effective mechanisms for the prevention of Kashin-Beck disease.

Objective To clone the fragment of hepatitis C virus(HCV) core gene and express it in E.coli.Methods The fragment of HCV core gene(approximate 366bp) was amplified by PCR and inserted into the pMD18-T vector.The cloned HCV core gene,which was confirmed by the digestion with EcoRⅠ/BamHⅠ,was subcloned into the expression vector pBV220 to construct recombinant plasmid pBV220/HCV-C.The expressed gene product was identified by SDS-PAGE and Western blotting.Results The fragment of HCV core gene was expressed successfully after temperature induction and a protein of 14 000 u was resulted.Conclusion Expression of the HCV-C gene in E.coli was achieved,which may be helpful for further studies on characterizations of HCV-C gene.

BACKGROUNDTo study the effect of gene amplification and selection system with DHFR plus GS and DHFR or GS gene on the foreign gene expression.

METHODSUsing the N-terminal truncated hTPO(T184) gene as target gene, two plasmidsre were constructed: pDC- T184 and pGC-T184 where DHFR and GS gene were used respectively as the selective amplification marker. They were cotransfected into CHO dhfr cells to establish dual gene amplification and selection system of DHFR plus GS gen and respectively transfected to establish single gene amplification and selection system of DHFR or GS gene. Three selective methods in dual selective system to compare expression efficiency of hTPO were designed: the first method (DG) was to use drug pressure of MTX, then use MSX; the second method (GD) was reversed; the third method was simultaneously to use MTX and MSX as drug pressure.

RESULTSDHFR+GS dual system had not only higher gene amplification efficiency but also higher level expression. There was no distinct affect in different method of drug pressure.

CONCLUSIONSMTX plus MSX dual drug pressure in dual selection system was an efficient and simple method to increase the expression of foreign gene in mammalian cells.

Animals ; CHO Cells ; Cricetinae ; Gene Amplification ; drug effects ; Gene Expression ; Glutamate-Ammonia Ligase ; genetics ; Methotrexate ; pharmacology ; Plasmids ; genetics ; Tetrahydrofolate Dehydrogenase ; genetics

BACKGROUNDTo explore the feasibility of cloning of the hepatocyte receptor interacting with the Pre S1 protein of HBV by two hybrid system.

METHODSYeast expression plasmids encoding fusion proteins of full length or portions of Pre S1 of HBV and DNA binding domain of yeast protein GAL4 were constructed and used to transform yeast reporter strain SFY526. Reporter gene product ?galactosidase activity was assayed as a measure of transcription activation in yeast. Mammalian expression plasmid encoding fusion proteins of full length Pre S1 and DNA binding domain of GAL4 was constructed and used to cotransfect hepatoma cell line Huh?7 together with CAT reporter plasmid. Cell extracts were assayed for CAT activity by thin?layer chromatography.

RESULTSThe fusion proteins of full length Pre S1 protein and GAL4 DNA binding domain present transcriptional activation function in yeast. The transcription activating sequence is localized to the 21 to 47 amino acids of Pre S1 protein Fusion proteins of full length Pre S 1 and GAL 4 DNA binding domain do not show transcriptional activation function in mammalian cells.

CONCLUSIONThe transcriptional activating sequence of HBVPre S1 protein in yeast overlaps the hepatocyte receptor binding site. The transcriptional activation function of HBV Pre S1 protein in yeast may prevent researchers?from using yeast two hybrid system to clone HBV receptor interacting with Pre S1 protein. However, the Pre S1 protein does not show transcriptional activation function in mammalian cells. Mammalian two?hybrid system may be a practical method to clone the HBV hepatocyte receptor interacting with Pre S1 protein.

DNA-Binding Proteins ; genetics ; Fungal Proteins ; genetics ; Hepatitis B Surface Antigens ; genetics ; Humans ; Protein Precursors ; genetics ; Recombinant Fusion Proteins ; genetics ; Transcriptional Activation ; Tumor Cells, Cultured ; Yeasts

Objective To investigate the relationship between Kashin-Beck Disease(KBD) and Human Parvovirus B19(HPVB19).Methods HPVB19DNA was detected in 55 sera of KBD patients and 52 healthy in adjacent non-endemic area and 35 healthy sera in normal area using PCR and then linked the HPVB19DNA to pGEM-T vector.The nucleotide sequence was analyzed and compared with HPVB19 nucleotide sequence published by Genebank and another in Journal of virology.Results HPVB19DNA was found in 16 of 55 sera in KBD patients,and the HPVB19DNA position rate(29.09%) is significantly higher than that of the two healthy control groups(11.54%、11.42% respectively)(P＜0.05).The nucleotide sequence homologies compared with the two published nucleotide sequence were 97.75%、97%,respectively.The putative amino acid homologies compared with the tow published were 93.5%.The amino acid variation was greater than the nucleotide sequence variation because of a base insertion.Conclusion There was a close relationship between HPVB19 infection and Kashin-Beck Disease.

Objective To investigate the feasibility of using synbiotics and probiotics to prevent and cure intestinal dysbacteriosis after burn. Methods Burned rats were fed with synbiotics and probiotics reagents, and the amounts of major intestinal florae in caecal contents were detected. Results The major physiological anaerobes were mostly stable, and the conditioned pathogens had no abnormal. Conclusion The micro-ecosystem regulator can quickly supplement the decreased physiological anaerobes caused by burning,and avoid the occurrence of dysbacteriosis.

Methylene blue injection has a stronger direct inactivation on VZV in vitro. When the injection was diluted from 1:16 to 1:128, which was obvious (P＜0.01 and P＜0.05). The MIC was 1:222, the IC50 was 1:135 and IC90 was 1:77. The results of microcellculture method showed when the injection was diluted from 1:16 to 1:64, it also effectively inhibited the proliferation of VZV in WISH continuous cell-lines (P＜0.01 and P＜0.05). The MIC was 1:95, the IC50 was 1:45 and the IC90 was 1:21.