Objective Getting the robust exogenous gene expression vector under the control of porcine insulin promoter, and to lay the foundation for pancreaticβ-cells specific transgene expressing pigs.Method Using porcine insu-lin promoter ( PIP, 1500 bp of the 5′UTR from the porcine INS gene including the first exon and the first intron) to con-struct expression vector, the HindIII restriction site which connected the sequences of PIP and EGFP was designed before ATG, named PIP-HindIII-EGFP.Considering that the different location of restriction site may affect the expression efficien-cy of the transgene, we optimized the expression vector.Firstly the HindIII restriction site was deleted to realize the seam-less connection of PIP and EGFP,the vector was named PIP-EGFP.Also we mutated the 3′intron splicing acceptor site( SA) of the first intron into HindIII restriction site, named as PIP-SA( M)-EGFP.Three different EGFP expression vectors were respectively transfected MIN-6 mouse pancreatic β-cells, pig ear fibroblasts and kidney cells.The transfected cells were cultured for 48 h and harvested for RT-PCR, flow cytometry and Western blot analysis, to analyze and compare the expres-sion efficiency of vectors.Results After transfection,green fluorescence was observed only in MIN-6 mouse pancreaticβ-cells.RT-PCR analysis and product sequencing showed that the three expression vectors did have different stability with in-tron splicing.The PIP-HindIII-EGFP construct and PIP-EGFP vector produced two kinds of mRNA with the first intron spliced and no spliced, indicating the instability of intron splicing.Mutation of the PIP splice site would cause the first in-tron not spliced, while flow cytometry and Western blot displayed that the mutation induced a most efficient expression of the downstream gene.Conclusions A robust and specific β-cells expression vector has been successfully generated by mutating the intron splicing acceptor site of the porcine insulin promoter.It provides the foundation for preparation of pigs with pancreaticβ-cells specifically expressing the transgene.

Atractylodis Macrocephalae Rhizoma and Atractylodis Rhizoma were widely used in strengthening spleen under different disease conditions, and were easily and often misused each other. Therefore, DNA barcode was used to distinguish Atractylodis Macrocephalae Rhizoma and Atractylodis Rhizoma from their adulterants to ensure the safe use. The sequence lengths of ITS2 of Atractylodes macrocephala, Atractylodis Rhizoma (A. lancea, A. japonica and A. coreana) were both 229 bp. Among the ITS2 sequences of A. macrocephala, only one G/C transversion was detected at site 98, and the average GC content was 69.42%. No variable site was detected in the ITS2 sequences of A. lancea. The maximum K2P intraspecific genetic distances of both A. japonica and A. coreana were 0.013. The maximum K2P intraspecific genetic distances of A. macrocephala, A. lancea, A. japonica and A. coreana were less than the minimum interspecific genetic distance of adulterants. The ITS2 sequences in each of these polytypic species were separated into pairs of divergent clusters in the NJ tree. DNA barcoding could be used as a fast and accurate identification method to distinguish Atractylodis Macrocephalae Rhizoma, Atractylodis Rhizoma, from their adulterants to ensure its safe use.
Atractylodes ; classification ; genetics ; DNA Barcoding, Taxonomic ; methods ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Drug Contamination ; prevention & control ; Drugs, Chinese Herbal ; chemistry ; classification ; Molecular Sequence Data ; Phylogeny ; Quality Control ; Rhizome ; classification ; genetics

Objective To analyze the literature distribution and research status of benign prostatic hyperplasia (BPH) in the elderly published in Chinese Medical Association journals.Methods The literatures of elderly BPH published in 88 kinds of Chinese Medical Association journals from 2009 to 2013 were collected.The publication year,distribution,content,author,institution and region of these literatures were analyzed.Results From 2009 to 2013,203 articles about elderly BPH were published in 10 kinds of journals.These literatures were published mostly in 2013 (50 articles,24.6％),secondly in 2010 (46 articles,22.7％) and 2009 (43 articles,21.2％),which were mostly published in Chinese Journal of Geriatrics (87 articles,42.9 ％).Most researches were focused on the fields of clinical treatment of BPH in the elderly,etiology,cross-sectional investigation and perioperative nursing,while the progresses in pathophysiology and diagnosis were relatively small,and the most published form was treatise (166 articles,81.8％).Authors who published the most articles were Yang Yong from Department of Urology Surgery in PLA General Hospital and Zhang Xianghua from Department of Urology Surgery in Peking University First Hospital (both,4 articles,3.5 ％),the institutions which published the most articles were Beijing Hospital and Beijing Chaoyang Hospital (both,9 articles,4.4 ％),and the provinces or cities which published the most articles were Beijing (75 articles,36.9％),Shanghai and Zhejiang (both,17 articles,8.4％) and Hunan (16 articles,7.9％).Among the 203 articles,35 articles (17.2％) were supported by founds.Conclusions Chinese Medical Association journals are the important sonrce to acquire literatures on elderly BPH for medical workers.The distribution of journals is relatively concentrated,but the distribution of author,institution and region in these articles is dispersed.Studies on clinical treatment of elderly BPH,etiology,inventory survey and perioperative nursing are especially concerned within nearly 5 years.

OBJECTIVETo investigate the effect of phosphorylated-ERK1/2 (p-ERK1/2) on inducible nitric oxide synthase (iNOS) expression in the substantia nigra (SN) of a mouse model of Parkinson's disease (PD), and explore the possible mechanism of dopaminergic (DA) neuron loss in the SN of the midbrain in PD.

METHODSPD was induced by intraperitoneal injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP) in C57BL/6N mice, and the behavioral changes of the PD mouse model were observed. Immunohistochemistry and Western blotting were used to detect the number of positive cells and the expressions of tyrosine hydroxylase (TH), p-ERK1/2 and iNOS in the SN of the PD mice, and their changes following Rg1 treatment were assessed.

RESULTSThe PD mice exhibited typical symptoms of PD, in which the number of TH-positive neurons and TH expression were significantly reduced by about 77% and 75% (P<0.01), respectively, 7 days after the 5th injection of MPTP as compared with those in the control group. Rg1 pretreatment significantly decreased the number of TH-positive neurons and TH expression by 44% and 41% (P<0.01), respectively. p-ERK1/2 expression was not observed in the cell nuclei until 1.5 h after the third injection of MPTP, and increased markedly at 6 h. Rg1 pretreatment significantly inhibited the expression of p-ERK1/2 and iNOS (P<0.01). A significant positive correlation was noted between the expression of p-ERK1/2 and iNOS (P<0.01).

CONCLUSIONP-ERK1/2 may regulate the expression of iNOS to induce DA neuron loss in the SN of PD, and Rg1 may protect the DA neurons possibly by depressing nuclear translocation of P-ERK1/2.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; Animals ; Extracellular Signal-Regulated MAP Kinases ; chemistry ; pharmacology ; Ginsenosides ; pharmacology ; Male ; Mice ; Mice, Inbred C57BL ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Parkinson Disease, Secondary ; chemically induced ; enzymology ; Phosphorylation ; Substantia Nigra ; enzymology

Objective To study interleukin-23 (IL-23) levels in serum and dendritic cells of acute-on-chronic liver failure (ACLF) patients with chronic hepatitis B (CHB) and to explore its relationship with the prognosis.Methods Peripheral blood mononuclear cells and serum were collected from 40 ACLF patients with CHB (including survival group 27 cases and non-survival group 13 cases) and 26 healthy controls.Monocytes were induced to immature dendritic cell in vitro and TNF-α was added to induce dendritic cell maturation.IL-23 mRNA of dendritic cells was detected by real time polymerase chain reaction (PCR), and serum IL-23 level was measured by enzyme-linked immuno sorbent assay (ELISA).Differences among the parameters with normal distribution were compared using t test, those with non-normal distribution were compared using non-parametric Mann-Whitney U-test, and the relationship between two variables was assessed by Spearman′s rank correlation.Results International normalized rate (INR) and model for end-stage liver disense (MELD) scores in non-survival group of ACLF were higher than those in survival group (INR: 2.32 vs 1.64, U=69.00, P=0.002 2;MELD:36 vs 30, U=64.50, P=0.001 4).However, there were no significant differences between two groups at gender, age, alanin aminotransferase (ALT), aspartate aminotrans ferase (AST), bilirubin, creatinine, hepatitis B virus (HBV) DNA, hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and serum IL-23.IL-23 mRNA level in dendritic cells at baseline in non-survival group of ACLF was significantly higher than that in survival group (76 vs 43, U=71.50, P=0.002 8).After treatment, serum IL-23 was significantly declined in survival group ([160±75] ng/L vs [91±49] ng/L, t=4.012, P=0.000 2), but not in non-survival group.Significant positive correlation was observed between IL-23 mRNA level in dendritic cells and MELD score at baseline (r=0.7198,P<0.01).Conclusions Persistent high serum IL-23 level suggests poor prognosis in ACLF patients with CHB.IL-23 mRNA expression in dendritic cells has good consistency with MELD score and the patients with high IL-23 mRNA expression has poor outcome.

Objective To investigate the association between the minisatellite polymorphism in the first exon of PLA2G4C gene and schizophrenia, and to reveal the important role of DNA sequence polymorphism in the pathogenesis of schizophrenia.Methods The minisatellite polymorphisms in the first exon of PLA2G4C gene in 91 patients with schizophrenia (case group)and 81 healthy persons (control group)were detected with PCR-sequencing analysis.The chi-square (χ2 )goodness-of-fit test was used to analyze the distribution of the PLA2G4C minisatellite polymorphism in various groups and to explore the association between the minisatellite polymorphism in the first exon of PLA2G4C gene and schizophrenia. Results There were minisatellite polymorphisms in PLA2G4C gene.Three kinds of polymorphisms 1×27 bp,2×27 bp and 3×27 bp were found by sequencing.The distribution of allelic frequencies at PLA2G4C polymorphism showed no statistical significance between case group and control group (P>0.05 ). No statistically significant difference was found in 3-homozygous haplotypes in PLA2G4C gene between case group and control group (P>0.05).At the same time,there was no statistically significant difference between 3-heterozygous haplotypes in PLA2G4C gene between case group and control group (P>0.05).Conclusion The minisatellite polymorphisms in the first exon of PLA2G4C gene are found,but the minisatellite polymorphism in the first exon of PLA2G4C gene may be not associated with the occurrence of schizophrenia.

BACKGROUND: Children usually exhibit pain-related behavior in the postanesthetic care unit. The aim of the present study was to compare the recovery and emergence profiles of children who received sevoflurane with caudal block or IV ketolorac or none for inguinal herniorrhaphy. METHODS: Forty five children, ASA 1, scheduled for herniorrhaphy were randomly assigned to receive either caudal block(n = 15), IV ketorolac (n = 15), or none (n = 15). All children were premedicated with midazolam(0.05 mg/kg) and glycopyrrolate (0.004 mg/kg) an hour before anesthesia induction. Thiopental sodium (5 mg/kg) and mask inhalation of sevoflurane 2 vol% in N2O/O2 50/50 were used to induce anesthesia. After induction, group 1 received none, while groups 2 and 3 received a caudal block and IV ketorolac, respectively. Anesthesia was maintained by sevoflurane with N2O/O2 inhalation via an endotracheal tube. Recovery was assessed by an independent observer using a postansthetic recovery score. Pain score was also assessed by an independent observer using a pain/discomfort scale. Recovery and agitation characteristics on emergence were compared between the three groups. RESULTS: There were no difference between the groups with respect to age, weight, duration of inhalation exposure, or recovery score. Agitation and pain scores were less in both the caudal block and IV ketorolac groups (P <0.05). Emergence delirium occurred less frequently in the caudal block and IV ketorolac groups (P <0.05). There was no significant difference between the caudal block and the IV ketorolac groups in emergence delirium. CONCLUSIONS: Emergence delirium after sevoflurane anesthesia was less common in the caudal block and IV ketorolac groups. Thus, it is presumed that the postoperative analgesic actions of caudal block or IV ketorolac reduce emergence delirium during recovery from sevoflurane anesthesia.
Anesthesia* ; Child* ; Delirium* ; Dihydroergotamine ; Glycopyrrolate ; Herniorrhaphy ; Humans ; Inhalation ; Inhalation Exposure ; Ketorolac* ; Masks ; Thiopental

The synthetic gene with 1640bp encoding for the anti-CD3/anti-CD20 bispecific single-chain antibody was designed and obtained by SOE (splicing by overlap extension) PCR. The cDNA was cloned into Flp-In expression vector pcDNA5/FRT and transfeced into Flp-In CHO cells to generate a stable expression cell line with a capacity for expressing anti-CD3/anti-CD20 bispecific single-chain antibody at 300 microg/L. The protein, which had a molecular weight of about 70 kD,was purified by Ni-NTA affinity chromatography and identified by SDS-PAGE and Western-blot analysis. Immunofluorescence assay and cellular rosetting showed that it can react specifically on Jurkat (CD3+) and Ramous (CD20+) cells. The lysis of human PBL against CD20-positive lymphoma Ramous cells in the presence of the anti-CD3/anti-CD20 bispecific single-chain antibody can observed by microscope. All these results would lighten the further study of its biological functions in vitro and in vivo.
Animals ; Antibodies, Bispecific ; biosynthesis ; genetics ; immunology ; Antibodies, Monoclonal ; immunology ; Antigens, CD20 ; immunology ; CD3 Complex ; immunology ; CHO Cells ; Cricetinae ; Cricetulus ; Humans ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Lymphoma, B-Cell ; metabolism ; Recombinant Proteins ; Transfection ; Tumor Cells, Cultured

AIMTo investigate the antioxidant mechanisms of propylene glycol mannate sulfate (PGMS) in hyperlipidemic rats.

METHODSMale Wistar rats were given high lipid emulsion diet to establish hyperlipidemic model. PGMS was given every day at different doses (37.8 and 75.6 mg.kg-1, ig) to hyperlipidemic rats for three weeks. In addition, diethyldithiocarbamate (DDC) was given 200 mg.kg-1.3 d-1 (i.p.) to inhibit SOD activity. Then, the MDA content was examined using TBA method to show the oxidation level, and the activities of SOD, GSH-Px and CAT were examined following the kit protocols to indicate the capability of eliminating OFR. RT-PCR was applied to study the expression of Cu, Zn-SOD mRNA in rat liver.

RESULTSThe MDA content of PGMS treatment groups decreased markedly compared with hyperlipidemic group, and the activities of SOD, GSH-Px and CAT increased distinctly. Cu, Zn-SOD mRNA expression was significantly increased by PGMS treatment. Furthermore, the application of DDC(the SOD inhibitor) reduced total SOD activity and Cu, Zn-SOD mRNA expression induced by PGMS, and the content of MDA increased correspondingly.

CONCLUSIONPGMS can induce the activities of antioxidant enzymes and the mRNA expression of Cu, Zn-SOD, which contribute to the elimination of oxygen free radical. This may explain the molecular mechanism of antioxidant effects of PGMS.

Animals ; Antioxidants ; pharmacology ; Catalase ; metabolism ; Free Radical Scavengers ; pharmacology ; Hyperlipidemias ; enzymology ; metabolism ; Liver ; enzymology ; metabolism ; Male ; Malondialdehyde ; metabolism ; Propylene Glycols ; pharmacology ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar ; Superoxide Dismutase ; biosynthesis ; genetics ; metabolism

Objective To investigate the charateristics of neuroendoscopic anatomy of the pineal region,discuss the anatomy passage and surgical tips of 2 different procedures,and provide anatomical application fundamentals for neuroendoscope-assisted operation of the pineal region.Methods Fifteen specimens of Chinese adult cadaveric head were selected randomly to simulate neuroendoscope-assisted microneurosurgery via suboccipital transtentorial and infratentorial supracerebellar routes.The exposure range and endoscopic adjustable degrees of anatomical structure of the pineal region through different routes were obsevred,and the related data of the anatomical structure via different routes were measured.Results In the simulating operation through suboccipital transtentorial route,the regulation of neuroendoscopy was flexible and both lateral anatomical structures of the pineal region were exposed clearly; in the simulating operation through inffatentorial supracerebellar route,inferior colliculis were not exposed clearly and the view of this route was stiffness and fixed with difficult regulation of the endoscope.The lengh of Galen's vein (GV) is (10.2±4.1) mm,that of internal cerebral vein (ICV) is (35.0±2.8) mm,that of basal vein (BV) is (35.4±6.4) mm and that of straight sinus is (51.1 ±5.2) mm; the angle GV and straight sinus formed (75.20±10.4)°,including acute angle in 14 cases and obtuse angle in 1.The imaginary extension of straight sinus located inferiorly to the splenium of corpus callosum was observed in 3 cases,located tangently to the splenium of corpus callosum in 11 cases,and located superiorly to the splenium of corpus callosum in 1 case.Conclusion Anatomic structures of the pineal region via neuroendosopic suboccipital transtentorial approach are exposed clearly than those via infratentorial supracerebellar approach,however,dissection oftentorium can improve the regulation ofneuroendoscope and exposition ofcontralateral anatomic structure.