OBJECTIVE: To study the chemical constituents from the ripe fruits of Cornus officinalis Sieb. et Zucc., and evaluate their protection on PC12 cells exposed to oxygen and glucose deprivation. METHODS: The compounds were isolated through various chromatographic methods including macroporous adsorption resin, silica gel, ODS, Sephadex LH-20 column chromatography, and preparative HPLC, and their structures were determined through spectroscopic analysis, including 1D and 2D NMR and MS data. RESULTS: Eight compounds were isolated from the water extract of the ripe fruits of Cornus officinalis Sieb. et Zucc., and their structures were elucidated as 6'-O-acetyl-7β-O-ethyl morroniside (1), chrysoderol(2), luteolin(3), 5-hydroxymethylfurfural(4), syringate(5),p-hydroxybenzoic acid(6), caffeic acid methyl ester(7), ethyl gallate(8). CONCLUSION: Compound 1 is a new iridoid glucoside, and compounds 2, 3, 5, 7 are obtained from Cornus officinalis for the first time. The MTT results show that compound 4 moderately increases the viability of OGD/R treated PC12 cells at the concentration of 1.0 μmol·L-1.

β-Amyrin synthase (β-AS) genes of Glycyrrhiza uralensis from 6 different regions were analyzed by PCR-SSCP and sequenced, then the correlationship between β-AS SNP and regions of Glycyrrhiza uralensis were determined. According to the 1 coding single nucleotide polymorphism on the first exon of β-AS gene at 94 bp site, Glycyrrhiza uralensis could be divided into 3 genotypes. In these genotypes, the percentage of 94A type in genuine regions was much higher, and it had significant differences with the percentage in non-genuine regions (P < 0.001). The results of the experiment proved that different β-AS genotypes at 94 bp site from different regions may be one of the important reasons to result in the genuineness of Glycyrrhiza uralensis.
Exons ; Genotype ; Glycyrrhiza uralensis ; classification ; enzymology ; genetics ; Intramolecular Transferases ; genetics ; Plant Proteins ; genetics ; Polymorphism, Single Nucleotide ; Polymorphism, Single-Stranded Conformational

OBJECTIVETo study the proliferation and differentiation of neural stem cells in the subventricular zone (SVZ) in neonatal rats after bilateral common arteries occlusion.

METHODSNinety-six 3-day-old Sparuge-Dawley rats were randomly divided into two groups: ischemia and control. Rats in the ischemia group were subjected to bilateral common arteries occlusion and the rats in the control group were sham-operated. All rats were administrated with 5-bromodeoxyuridine (BrdU) (50 mg/kg) via intraperitoneal injection. Rats were sacrificed and their brains were removed 1, 4, 7, 10, 14 and 35 days after ischemia. Using brain paraffin sections and immunofluorescence assays, the number of newborn cells in the SVZ was counted. Newborn neural stem cells and oligodendrocytes in the SVZ were observed, and then double marked with BrdU and nestin or osmium tetroxide (O4).

RESULTSThe number of BrdU+ cells (neural stem cells) in the SVZ in the ischemia group was greater than in the control group 4, 7, 10 and 14 days after ischemia, and reached a peak at 4 days after ischemia (253.1+/- 49.3 vs 133.5+/- 17.7; P< 0.01). By 35 days after ischemia, the number of BrdU+/O4+ cells (oligodendrocytes) in the corpus callosum (56.0+/- 7.2 vs 17.0+/- 6.4; P< 0.01), the septal nuclei (45.0+/- 11.9 vs 20.5+/- 5.0; P< 0.01), the striatum (34.5+/- 4.2 vs 14.5+/- 5.8; P< 0.01) and the olfactory bulb (46.5+/- 6.6 vs 23.5+/- 8.4; P< 0.01) in the ischemia group increased significantly as compared to the control group (P< 0.01).

CONCLUSIONSBrain ischemia can activate the proliferation of neural stem cells in the SVZ and promote neural stem cells differentiation into oligodendrocytes. The immature brain may have the capacity for self-repair after ischemic brain injury.

Animals ; Animals, Newborn ; Brain Ischemia ; physiopathology ; therapy ; Bromodeoxyuridine ; metabolism ; Cell Differentiation ; Cell Proliferation ; Cerebral Ventricles ; physiopathology ; Female ; Male ; Neurogenesis ; Rats ; Rats, Sprague-Dawley ; Stem Cell Transplantation

OBJECTIVETo investigate the effects of formaldehyde inhalation on the morphological damage, and Glu, GABA and NOS contents in olfactory bulb and hippocampus of rats.

METHODSTwenty SD rats were equally divided into two groups: rats in the control group inhaled fresh air, while the animals in experimental group were exposed to the air containing formaldehyde (12.5 mg/m(3), 4 h/d) for 7 days. Then rats were sacrificed and frozen sections of olfactory bulb and hippocampus were prepared. The morphological changes were examined and the Glu, GABA and NOS contents were detected using Nissl-staining, immunohistochemistry and Western blot, respectively.

RESULTCompared with the control group, there was a significant confusion and shrink of neuron morphology in experimental group, the number and staining intensity of Glu and NOS positive cells and protein contents were reduced. The protein expression of GABA was also decreased in the formaldehyde group.

CONCLUSIONFormaldehyde inhalation can cause a severe morphological damage of olfactory bulb and hippocampus in SD rats,which may further impair memory and learning ability through the reduction of Glu, GABA and NOS expression.

Animals ; Formaldehyde ; toxicity ; Glutamic Acid ; metabolism ; Hippocampus ; drug effects ; metabolism ; pathology ; Inhalation Exposure ; Learning ; drug effects ; Neurons ; drug effects ; metabolism ; pathology ; Nitric Oxide Synthase ; metabolism ; Olfactory Bulb ; drug effects ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley ; gamma-Aminobutyric Acid ; metabolism

Objective To estimate the value of computed tomography perfusion for clinical stage and approach the correlation of perfusion parameters and Cyfra21-1.Methods 63 patients with head and neck squamous carcinoma were confirmed by pathology and follow up underwent CT perfusion,which were divided into three groups by international clinical staging criteria(stageⅠ,stageⅡand stageⅢ-Ⅳ).BF,BV,MTT,TTP and Cyfra21-1 were recorded and compared with correlation in different clinical staging.Results There was no significant difference of Cyfra21-1 between stageⅠand stageⅡ(Z =1.439,P =0.1 62).There was significant differ-ence of Cyfra21-1 between stageⅠand stageⅢ-Ⅳ(Z =3.356,P =0.000),stageⅡand stageⅢ-Ⅳ(Z =4.959,P =0.000).There was significant difference of BF and BV between stageⅠand stageⅡ,stageⅠand stageⅢ-Ⅳ(P <0.05),of MTT and TTP between stageⅠand stageⅢ-Ⅳ,stageⅡand stageⅢ-Ⅳ(P <0.05).There was no significant difference of BF and BV between stageⅡand stageⅢ-Ⅳ(P >0.05),of MTT and TTP between stageⅠand stageⅡ(P >0.05).Cyfra21-1 and perfusion parameters in all groups have relationship(r=0.76,0.76,-0.82,-0.82,P <0.05).Conclusion The statistically significant of positive correlation be-tween Cyfra21-1 and perfusion parameters in head and neck squamous carcinoma suggests that CT perfusion could play a complemen-tary role in clinical assessment.

Objective To re-evaluate the immunogenicity of meningococcal serogroups A and C polysaccharide vaccine among a healthy population of age 5 to 59.Methods Pre and post-vaccination ser-um samples were collected from the subjects involved in a randomized , controlled clinical trial conducted in 2005 at Hechi, Guangxi, China.Enzyme-linked immunosorbent assay (ELISA) was performed for the quan-titative detection of specific anti-capsule IgG antibody against meningococcal serogroups A and C in serum samples.Serum bactericidal assay ( SBA) was used to measure the bactericidal antibody activity against se-rougroups A and C bacterial strains .Results Geometric mean concentrations (GMCs) of specific anti-cap-sule IgG antibody were 23.66 μg/ml and 78.83 μg/ml for serogroup A (P<0.05), and 1.23 μg/ml and 51.25 μg/ml for serogroup C (P<0.05) in serum samples of pre and post-vaccination, respectively.Be-fore immunization , 99% of serum samples ( 97/98 ) showed serogroup A-specific IgG concentration ≥2μg/ml, which reached up to 100%(98/98) after vaccination (P>0.05).The percentage of serum sam-ples with serogroup C-specific IgG concentration ≥2 μg/ml rose from 20%to 99% after vaccination ( P<0.05).The ratio of positive serum samples with at least four times of increases in concentration of specific IgG against serogroup A and serogroup C after vaccine immunization were 34% and 100%, respectively . The rSBA geometric mean titers ( GMTs) for serogroups A and C in serum sample of post-vaccination were respectively elevated from 1164 to 5530 (P<0.05), and from 4 to 6225 (P<0.05).The percentage of se-rum samples with rSBA GMTs≥128 increased from 91% to 99% for serogroup A (P>0.05), and from 14%to 96%for serogroup C (P<0.05) after vaccination.The rSBA GMTs with at least four times of in-creases after vaccination were detected respectively in 53% and 100% of serogroups A and C vaccinated subjects.Pearson correlation coefficient between IgG concentration and rSBA GMTs was r=0.15 (pre) and r=0.23 (post) for serogroup A, and r=-0.14 (pre) and r=0.58 (post) for serogroup C (P<0.05). Conclusion This study has demonstrated an efficient and sustained immunogenicity with meningococcal sero -groups A and C polysaccharide vaccine as evidenced by the data from standardized assays of ELISA and SBA .

OBJECTIVETo investigate the effect of ERCC1 gene on the repair capability of damaged DNA in lung cancer A549 cells induced by benzo[a]pyrene.

METHODSRecombinant plasmid expressing ERCC1 antisense RNA was constructed and transfected into A549 cells by Lipofectin reagent. The stable-transfected cell colonies were selected by hygromycin. Cell viability was determined by the MTT assay. The level of ERCC1 mRNA was measured by Northern Blot analysis. Single cell gel electrophoresis assay was applied to determine the cellular DNA damage and fifty cells for each group were counted.

RESULTSSeven positive colonies expressing ERCC1 antisense RNA were screened. There was no growth rate difference between the antisense-transfected cells and the parental cells. The endogenous mRNA level in transfected colonies decreased in varied degrees, i.e. 12% approximately 86% of that of the parental cells in Northern Blot assay. After 24 h treatment of 10 micro mol/l benzo[a]pyrene, the repair capability for DNA damage in transfected colonies was reduced to 29% approximately 71% of that of the parental cells. Also, a statistically significant correlation was observed between expression of ERCC1 mRNA and repair capability (r = 0.84).

CONCLUSIONAntisense ERCC1 RNA decreased the repair capability for damaged DNA in lung cancer cells induced by benzo[a]pyrene.

Benzo(a)pyrene ; toxicity ; Cell Line, Tumor ; DNA Damage ; drug effects ; DNA Repair ; drug effects ; DNA-Binding Proteins ; genetics ; metabolism ; pharmacology ; Endonucleases ; genetics ; metabolism ; pharmacology ; Humans ; Lung Neoplasms ; pathology ; Plasmids ; RNA, Antisense ; pharmacology ; RNA, Messenger ; metabolism ; Repressor Proteins ; Transfection

OBJECTIVETo study the expression levels of ERCC2, UDG, and PCNA in cisplatin-treated A549 cell line.

METHODComet assay, RT-PCR, and western blot were used to study the mRNA and protein expression levels of ERCC2, UDG, and PCNA.

RESULTSWhen treated with IC(20) cisplatin, the DNA damage level increased as the cisplatin treated time increased within 24 h of cisplatin treatment. The tail state 12 h and 24 h after treatment was 5.02 +/- 0.68 and 7.22 +/- 0.53 respectively, which was significantly higher than those of the controls (2.73 +/- 0.29). The tail state 24 h after treatment was not significantly different from that of the controls. The DNA damage level decreased to normal after cisplatin treatment in 24 h (tail state 3.64 +/- 0.7). The expression levels of ERCC2, UDG, PCNA protein (4.37 +/- 0.57, 5.47 +/- 0.46, 2.21 +/- 0.47 respectively) and mRNA (0.71 +/- 0.08, 0.74 +/- 0.06, 0.82 +/- 0.09) were increased significantly within 24 h exposure and decreased to normal 24 h after cisplatin treatment. The 3 enzymes' mRNA and protein expression increased when treated with cisplatin, but the changes of protein level were slower than those of mRNA levels.

CONCLUSIONSThe DNA repair capability in A549 cells increases after cisplatin treatment. Cisplatin was a positive regulation of ERCC2, UDG, PCNA expression levels, which causes the increase of mRNA, and protein. The positive regulation only works in a short time and returns normal after 24 h of cisplatin treatment.

Antineoplastic Agents ; pharmacology ; Biomarkers, Tumor ; Cell Line, Tumor ; Cisplatin ; pharmacology ; Comet Assay ; DNA Glycosylases ; biosynthesis ; genetics ; DNA Helicases ; DNA Repair ; drug effects ; DNA-Binding Proteins ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Proliferating Cell Nuclear Antigen ; biosynthesis ; genetics ; Protein Biosynthesis ; Proteins ; genetics ; RNA, Messenger ; biosynthesis ; Transcription Factors ; Xeroderma Pigmentosum Group D Protein

OBJECTIVETo construct T vectors based on Xcm I recognition site and optimize the PCR fragments for its ligation.

METHODSWe firstly cloned the human histone H4 cDNA containing one Xcm I recognition site at both its 5' and 3' end into pCDNA 3.0 vector and then generated T vector with pCDNA 3.0 backbone by cutting the recombinant plasmid with Xcm I. To increase the ligation efficiency, the primers were firstly phosphorylated before DNA fragments amplification and then the PCR products were treated with Taq DNA polymerase and dATP after PCR amplification. Two DNA fragments with the length of 312 bp and 1 329 bp were ligated to it and the ligation mixture was transformed into E. coli DH5α competent cells and the positive rates of the transformants were evaluated by PCR and DNA agarose gel electrophoresis.

RESULTSOur results showed that the T vector produced by our method could ligate to the target DNA fragments with high efficiency. Besides, the phosphorylation state of the primers used for PCR amplification is also an important factor determining the cloning efficiency. What's more, as for longer DNA fragments, retreatment with Taq DNA polymerase and dATP after PCR amplification and purification could improve the ligation efficiency significantly.

CONCLUSIONOur protocol may overcome the dependence on blue/white screening to get positive clones and provide a potent way to generate T vectors and ligate them to the target PCR fragment.

Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; Genetic Vectors ; Histones ; genetics ; Humans ; Polymerase Chain Reaction ; methods

OBJECTIVETo develop a quantitative method for methylation analysis of the p16 gene based on mismatch hybridization and chemiluminescence.

METHODSGenomic DNA was modified by sodium bisulfite to convert all unmethylated but not methylated cytosines to uracil, and subsequently a pair of primer having no CpG sites was designed for amplification target DNA containing methylated or unmethylated CpG sites. The PCR product spanning CpG sites were hybridized with two oligonucleotide probes which perfectly matched the methylated and unmethylated CpG sequences respectively, and the hybrids were detected by chemiluminescent method. The percentage of methylated target sequences could be estimated by calculating the ratio of signals obtained with two probes.

RESULTSThe percentage of methylation of artificial mixtures DNA showed a linear relation. There was a negative correlation between the methyaltion index with p16 transcriptional mRNA of p16 gene in tumor cell lines.

CONCLUSIONCompared with existing methods, this assay is nonisotopic, rapid, simple, and can be widely applied to the study of DNA methylation.

Cell Line, Tumor ; CpG Islands ; DNA Methylation ; Genes, p16 ; Humans ; Luminescent Measurements ; Nucleic Acid Hybridization ; Promoter Regions, Genetic ; Reverse Transcriptase Polymerase Chain Reaction ; Sulfites