A simple and effective method for the duplex PCR detection was developed by using sequences of exogenous gene(RDV MP-)and endogenous gene(Zein)as templates for PCR amplification.The results of routine PCR amplification for RDV MP-gene in transgenic maize suggested that RDV MP-gene can stably inheritate in transgenic plants and their progenies;The duplex PCR detection of all negative and part positive samples that obtained by routine PCR amplification confirmed that above negative results were exact,also showed that the quality of extracted DNA can meet the need of PCR amplification.The error ratio of negative samples was 1.4%.The method used in this study was simple and credible and can be used to detect transgenic plants and their products.

Objective:To investigate polymorphism of human leukocyte antigen (HLA)-DRB1 and -DQB1 genes in Bai ethnic group in Dali,Yunnan province.Methods:Polymerase chain reaction-sequence specific primers (PCR-SSP) were used to determine HLA-DRB1 and -DQB1 alleles in 124 unrelated healthy Bai ethnic individuals living in Eryuan County of the Dali Bai autonomous prefecture,Yunnan province.Results:Among all the 21 DRB1 alleles and 15 DQB1 alleles were identified,the predominant alleles were DRB1*1202(26.61%),DRB1*0901(13.89%) and DRB1*0803(9.92%) on DRB1 locus and DQB1*0301(31.45%),DQB1*0601(10.08%),DQB1*0401(8.06%)and DQB1*0502(8.06%)on DQB1 locus.The most common haplotypes were DRB1*1202-DQB1*0301(20.08%)and DRB1*0803-DQB1*0601(7.19%).Conclusion:The phylogenetic tree constructed according to the HLA-DRB1,-DQB1 allele frequencies of Bais with those of other 10 populations suggests that the Bai ethnic group belongs to the southern group of China,but it keeps genetic distance from others and the HLA genes exhibits a unique profile.This study would provide HLA polymorphism information of Bai for the future investigation on the disease related to the genetic polymorphism.

OBJECTIVETo demonstrate the effects and mechanisms of Huangkui capsule (HKC) on renal fibrosis in rats with diabetic nephropathy (DN).

METHODRats were randomly divided into 5 groups, the sham-operated group (Sham group, n = 5), the vehicle-given group (Vehicle group, n = 7), the low dose of HKC-treated group (L-HKC group, n = 7), the high dose of HKC-treated group (H-HKC group, n = 7) and the lipoic acid (LA)-treated group (LA group, n = 7). DN models were induced by intraperitoneal injection of streptozotocin (STZ,35 mg x kg(-1)) twice and unilateral nephrectomy. After models were successfully established, the rats in HKC and LA groups were daily administrated with HKC suspensions (0.75, 2 g x kg(-1)) or LA suspensions (60 mg x kg(-1)) respectively, and at the same time, the rats in Vehicle group were daily administrated with distilled water (2 mL) for 8 weeks. All rats were sacrificed at the end of week 8 to collect blood and renal tissues. UAlb, renal function, renal fibrotic morphologic characteristics, as well as oxidative stress (OS)-related markers, the protein expressions of the key signaling molecules in p38 mitogen-activated protein kinase (p38MAPK) signaling pathway, fibrogenic cytokines and inflammatory factors were examined respectively.

RESULTHKC, similar to LA, improved the general state of health, body weight, UAlb, BUN, UA and Alb in DN model rats. Of note, renal fibrosis was ameliorated in HKC groups,especially in H-HKC group which was better than that in LA group. In addition, HKC not only improved the main indexes of OS in the kidney like LA, but also down-regulated the protein expressions of phosphorylated-p38MAPK (p-p38MAPK), transforming growth factor (TGF)-β1 and tumor necrosis factor(TNF)-α in the kidney, whereas, LA only decreased the protein expression of TNF-α in the kidney in DN model rats.

CONCLUSIONHKC, similar to LA, has the actions of anti-OS in vivo. Moreover, HKC could attenuate renal fibrosis by suppressing the activation of p38MAPK signaling pathway and the protein expressions of fibrogenic cytokines and inflammatory factors in the kidney in DN model rats, which is different from LA.

Abelmoschus ; chemistry ; Animals ; Capsules ; Diabetic Nephropathies ; drug therapy ; metabolism ; pathology ; Drugs, Chinese Herbal ; pharmacology ; Fibrosis ; Kidney ; drug effects ; pathology ; MAP Kinase Signaling System ; drug effects ; Male ; Oxidative Stress ; drug effects ; Rats ; Rats, Sprague-Dawley ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors

In the development of diabetic nephropathy (DN), reactive oxygen specie (ROS) over much in vivo leads to oxidative stress(OS)-related renal injuries, which are characterized by the structural and functional changes in glomerular and renal tubular cells in morphology. The regulative approaches of OS involve the several signaling pathways, in which, both p38 mitogen-activated protein kinase (MAPK) signaling pathway and adenosine monophosphate-activated protein kinase (AMPK) signaling pathway play the important roles as the target of anti-oxidants. The interventional actions of Chinese herbal compound prescriptions and the extracts of single Chinese herbal medicine (CHM) on OS in the kidney in DN include regulating the balance between ROS and antioxidants, reducing the production of AGEs, inhibiting the expression of growth factors and intervening the activity of signaling pathways.
Animals ; Diabetic Nephropathies ; drug therapy ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Kidney ; drug effects ; metabolism ; Oxidative Stress ; drug effects ; Signal Transduction ; drug effects ; Treatment Outcome

Objective To investigate the association and difference between patent foramen ovale(PFO), atrial septal aneurysm(ASA), atrial septal defect(ASD) in normal controls and cryptogenic ischemic stroke(CS) in youth diagnosed by double source CT.Methods A total of 168 CS patients and 180 controls matched age and gender were included in the present study.The two groups were diagnosed by double source CT and clinical materials.The incident rate of PFO, ASA, ASD, the degrees of PFO, ASD,the lengths of PFO and difference between CS in two groups were analysed.Results The incident rates of PFO, ASA, ASD were 40.6%,10.7%,6.5% and 15.6%,3.3%,2.2% in CS groups and controls respectively(P<0.001).The incident rates of PFO merged ASA and ASA merged ASD were 6.0%,3.0% and 0%,0% respectively between two groups(P<0.001).The diameters of PFO, ASD were (1.71±0.62) mm,(3.42±0.72) mm and (0.85±0.51) mm,(2.45±0.42) mm in two groups respectively(P<0.001).The lengths of PFO were (14.6±3.8) mm and (8.2±2.3) mm in two groups(P<0.001).The correlations between stroke in two groups were no difference(P>0.001).Conclusion PFO,ASA and ASD are important to CS.While PFO,ASA and ASD can accurately be diagnosed by double source CT.

OBJECTIVETo explore the DNA methylation levels of genome in cFb transdifferentiation induced by SiO2 in rats.

METHODSThe primary macrophages and fibrocytes of SD rats were co-cultured directly and indirectly, which were exposed to SiO2 at the doses of 25, 50 and 100 g/ml. The transdifferentiation of cFb was identified with immunohistochemical assay. The genomic DNA methylation levels of cFb were detected with HPLC.

RESULTSUnder the condition of indirect co-culture, as compared with control group, the genomic DNA methylation levels of cFb exposed to SiO2 at the doses of 25, 50 and 100 g/ml reduced by 19.9%, 26.9% and 30.3%, respectively (P < 0.05); as compared with cFb exposed to 100 g/ml SiO2, the genomic DNA methylation levels of cFb exposed to 5-aza-dC decreased by 22.0% (P < 0.05). Under the condition of ThinCert(TM) direct co-culture, as compared with control group, the genomic DNA methylation levels of cFb exposed to SiO2 at the doses of 25, 50 and 100 g/ml reduced by 22.2%, 30.2% and 36.7%, respectively (P < 0.05); as compared with cFb exposed to 100 g/ml SiO2, the genomic DNA methylation levels of cFb exposed to 5-aza-dC decreased by 20.6% (P < 0.05).

CONCLUSIONUnder the co-culture condition in vitro, SiO2 could reduce the genomic DNA methylation levels of cFb. The ThinCert(TM) direct co-culture can be used to study the silicosis fibrosis.

Animals ; Cell Transdifferentiation ; drug effects ; Cells, Cultured ; Coculture Techniques ; DNA Methylation ; Fibroblasts ; cytology ; drug effects ; Genome ; drug effects ; Lung ; cytology ; drug effects ; Male ; Rats ; Rats, Sprague-Dawley ; Silicon Dioxide ; adverse effects

Objective: To assess the risk of public health emergencies in Zhejiang Province, March 2021. Methods: An expert counsel was conducted to assess the risk of coronavirus disease 2019 ( COVID-19 ) , enteritis due to norovirus, chicken pox and influenza by professionals in Zhejiang CDC, based on the information from infectious disease and public health emergency surveillance in Zhejiang Province, domestic health administrative departments, World Health Organization, and European CDC. Results: In March 2021, the risk of imported COVID-19 epidemic will be high in Zhejiang Province, and the possibility of local spread could not be ruled out. The possibility of a large-scale outbreak of enteritis due to norovirus and a small-scale outbreak of chickenpox in schools and kindergartens could not be ruled out after the new term begins. An increased risk of influenza epidemic is predicted in collective units such as schools and kindergartens, yet the risk of a large-scale one will be low. Conclusion High attention should be paid to COVID-19 and enteritis due to norovirus, and general attention should be paid to chicken pox and influenza outbreak.

High concentration of corticosterone leads to morphological and functional impairments in hippocampus, ranging from a reversible atrophy of pyramidal CA3 apical dendrites to the impairment of long-term potentiation (LTP) and hippocampus-dependent learning and memory. Glutamate and N-methyl-D-aspartate (NMDA) receptor play an important role in this effect. Because of the importance of brain-derived neurotrophic factor (BDNF) in the functions of the hippocampal neurons, alteration of the expression of BDNF is thought to be involved in the corticosterone effect on the hippocampus. To determine whether change in BDNF in the hippocampus is involved in the corticosterone effect, we injected corticosterone (2 mg/kg, s.c.) to Sprague-Dawley rats and measured the mRNA, proBDNF and mature BDNF protein levels in the hippocampus. We also measured the phosphorylation level of the transcription factor cAMP response element binding protein (CREB). Furthermore, we intraperitoneally injected NMDA receptor antagonist MK801 (0.1 mg/kg) 30 min before corticosterone administration to investigate whether and how MK801 affected the regulation of BDNF gene expression by corticosterone. Our results showed that 3 h after single s.c. injection of corticsterone, the expression of BDNF mRNA, proBDNF and mature BDNF protein decreased significantly (P<0.01). MK801 promoted the downregulation of BDNF gene expression in the rat hippocampus by corticosterone. We also found that either applying corticosterone or co-applying corticosterone with MK801 downregulated the phosphoration level of CREB, the latter (corticosterone plus MK801) being more effective (P<0.05). Taken together, our results indicate that corticosterone downregulates BDNF gene expression in the rat hippocampus through CREB pathway and that blockade of NMDA receptor enhances this effect of corticosterone in reducing BDNF expression.
Animals ; Brain-Derived Neurotrophic Factor ; genetics ; metabolism ; Corticosterone ; pharmacology ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Dizocilpine Maleate ; pharmacology ; Down-Regulation ; drug effects ; Hippocampus ; metabolism ; Male ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, N-Methyl-D-Aspartate ; antagonists & inhibitors ; Signal Transduction ; drug effects

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