Transcriptomic Analysis of Wuzi Yanzongwan on Testicular Spermatogenic Function in Semi-castrated Male Mice / 中国实验方剂学杂志

ObjectiveTo screen out the transcriptomes related to the intervention of Wuzi Yanzongwan on the spermatogenic function of semi-castrated male mice， and to explore its potential mechanism in the intervention of the progress of low spermatogenic function. MethodBalb/c mice were randomly divided into sham-operated group， model group， testosterone propionate group（0.2 mg·kg-1·d-1， intramuscular injection） and Wuzi Yanzongwan group（1.56 g·kg-1·d-1， intragastric administration） according to body weight， with 12 mice in each group. The right testicle and epididymis were extracted from the model group and the drug administration group to construct the semi-castrated model of low spermatogenic function， while the fur and the right scrotum of the sham-operated group were only cut and immediately sterilized and sutured. At the end of the intervention， hematoxylin-eosin（HE） staining was used to observe the histopathology of testis， enzyme-linked immunosorbent assay（ELISA） was used to detect the levels of serum testosterone（T）， luteinizing hormone（LH） and follicle stimulating hormone（FSH）. The sperm count and motility of epididymis were measured by automatic sperm detector of small animal. Transcriptomic microarray technology was used to detect the mRNA expression level of testicular tissue in each group， the transcriptome of genes related to the regulation of Wuzi Yanzongwan was screened， and three mRNAs were selected for Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） to verify the transcriptome data. Through the annotation analysis of Gene Ontology（GO） and the signaling pathway analysis of Kyoto Encyclopedia of Genes and Genomes（KEGG）， the related functions of drugs regulating transcriptome were analyzed. ResultCompared with the sham-operated group， the testicular tissue of mice in the model group showed spermatogenic injury， contraction and vacuolization of the seminiferous tubules， reduction of spermatogenic cells at all levels， widening of the interstitial space， obstruction of spermatogonial cell development and other morphological abnormalities， and serum T significantly decreased， LH significantly increased（P<0.01）， and FSH elevated but no statistically significant difference， the count and vitality of epididymal sperm significantly decreased（P<0.01）. There were 882 differentially expressed mRNAs in the testicular tissues， of which 565 were up-regulated and 317 were down-regulated. Cluster analysis showed that these differentially expressed mRNA could effectively distinguish between the sham-operated group and the model group. Compared with the model group， the damage to testicular tissue in the Wuzi Yanzongwan group was reduced， the structure of the seminiferous tubules was intact， vacuolization was reduced， and the number of spermatogenic cells at all levels was significantly increased and arranged tightly. The serum T significantly increased， LH significantly decreased（P<0.01）， and FSH decreased but the difference was not statistically significant. The count and vitality of sperm in the epididymis were significantly increased（P<0.01）. Moreover， Wuzi Yanzongwan could regulate 159 mRNA levels in the testes of semi-castrated mice， of which 32 were up-regulated and 127 were down-regulated， and the data of the transcriptome assay was verified to be reliable by Real-time PCR. GO and KEGG analysis showed that the transcriptome functions regulated by Wuzi Yanzongwan were involved in the whole cell cycle process of sperm development such as sex hormone production of interstitial cells in testis， renewal， differentiation， metabolism， apoptosis and signal transduction of spermatogenic cells， and were closely related to the biological behaviors of signaling pathways such as spermatogenic stem cell function， endoplasmic reticulum protein processing and metabolic program. ConclusionWuzi Yanzongwan can effectively improve the low spermatogenic function of semi-castrated male mice， and its mechanism may be related to the regulation of testicular transcriptional regulatory network， the synthesis of sex hormones in testicular interstitial cells， the function of spermatogenic stem cells， the whole cell cycle process of spermatogenesis， as well as the expression of endoplasmic reticulum protein processing and metabolic program related genes transcription.

Screening of Reference Genes of Andrographis paniculata Under MeJA and Abiotic Stresses by Real-time Fluorescence-based Quantitative PCR / 中国实验方剂学杂志

ObjectiveTo screen the appropriate reference genes for real-time fluorescence-based quantitative polymerase chain reaction（Real-time PCR）analysis of the Andrographis paniculata under methyl jasmonate（MeJA）and various abiotic stresses. MethodThe actin 1（ACT1），actin 2（ACT2），elongation factor（EF-1α），glyceraldehyde-3-phosphate dehydrogenase（GAPDH），tubulin（TUB），polyubiquitin（UBQ）， and 18S rRNA（18S）gene were selected as candidate reference genes based on the RNA-seq data of high temperature，drought， UV， and MeJA. The expression of seven candidate reference genes in the A. paniculata leaves was assessed by Real-time PCR，and the stability was analyzed by geNorm，NormFinder，BestKeeper， and Refinder. ResultThe results of stability evaluated by geNorm，NormFinder， and BestKeeper were not the same due to different indicators. As analyzed by Refinder， for the stability of the expression， the genes were ranked as UBQ>18S>EF-1α>ACT2>ACT1>GAPDH>TUB under high temperature stress， ACT1>UBQ>EF-1α>18S>ACT2>GAPDH>TUB under drought stress， EF-1α>TUB>ACT2>UBQ>18S>GAPDH>ACT1 under UV stress， and ACT1>EF-1α>UBQ>ACT2>18S>TUB>GAPDH under MeJA stress. Among them，18S gene was not suitable as an internal reference gene duo to its high expressive abundance. This study also verified the relative expression level of andrographolide synthesis-related gene hydroxy-methylglutaryl-CoA synthase （HMGS） in the four stresses on the basis of transcriptome data，and found that the Real-time PCR results of appropriate internal reference genes were accurate and reliable. ConclusionUBQ-ACT1-UBQ，EF-1α-TUB，and ACT1-EF-1α were the suitable combinations under stresses of high temperature，drought，UV， and MeJA. This study is expected to provide references for the research on function regulation and expression of genes in A. paniculata under high temperature，drought，UV， and MeJA stresses.

Cloning and Prokaryotic Expression of Glycosyltransferase Genes from Paris polyphylla var. yunnanensis / 中国实验方剂学杂志

Objective:To clone the full-length glycosyltransferase genes （<italic>PpUGT</italic>1，<italic>PpUGT</italic>7） related to saponins biosynthesis in <italic>Paris polyphylla</italic> var. <italic>yunnanensis</italic>，and perform bioinformatics analysis，relative expression analysis and prokaryotic expression analysis. Method:Total RNA was isolated from <italic>P. polyphylla </italic>var. <italic>yunnanensis </italic>with use of the Eastep^® Super Total RNA Extraction Kit and converted to cDNA. Specific primers were designed according to the transcriptome data to clone the full-length gene. Relevant software was then used for bioinformatic analysis of the protein sequences. The relative gene expression levels were detected by real-time fluorescent quantitative polymerase chain reaction （Real-time PCR） and the prokaryotic expression vectors were built to heterologously express recombinant protein in <italic>Escherichia coli.</italic> Result:The open reading frame （ORF） of <italic>PpUGT</italic>1 was 1 827 bp，encoding 608 amino acids，and was predicted as a steroid glycosyltransferase；the ORF of <italic>PpUGT</italic>7 was 1 380 bp，encoding 459 amino acids，and was predicted as a triterpenoid glycosyltransferase. The calculated relative molecular mass of two proteins were 67.6 kDa and 51.3 kDa respectively，and both of them were hydrophilic proteins，no transmembrane domain，no signal peptides，both showing high similarity and conservativeness with homologous sequences. The results of Real-time PCR showed that the expression level of <italic>PpUGT</italic>1 was root>leaf>flower>stem；the expression level of <italic>PpUGT</italic>7 was stem>leaf>flower>root. In addition，PpUGTs proteins were expressed in <italic>E. coli</italic>. in a soluble form. Conclusion:The genes of <italic>PpUGT</italic>1 and <italic>PpUGT</italic>7 were cloned successfully. Real-time PCR showed the genes were expressed differently in different plant organs， and their recombinant proteins were successfully expressed in <italic>Escherichia coli</italic>. This study lays a foundation for functional characterization of PpUGTs and analysis of the biosynthesis pathway of saponins in <italic>Paris polyphylla </italic>var. <italic>yunnanensis</italic>.

Usefulness of Real-time PCR to Detect Mycobacterium tuberculosis and Nontuberculous Mycobacteria / 결핵및호흡기질환

BACKGROUND: The purpose of this study was to evaluate recently developed real-time polymerase chain reaction (PCR) assay kit to detect Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) in respiratory specimens. METHODS: We assessed the positive rate of the real-time PCR assay to detect MTB and NTM in 87 culture-positive specimens (37 sputum, 50 bronchial washing), which were performed real-time PCR by using Real-Q(TM) MTB&NTM Kit from January 2009 to June 2009, at Gyeongsang University Hospital. To compare the efficacy with the TB-PCR assay, we evaluated 63 culture-positive specimens (19 sputum, 44 bronchial washing) for MTB or NTM, which were performed TB-PCR by using ABSOLUTE(TM) MTB II PCR Kit from March 2008 to August 2008. RESULTS: Among 87 specimens tested using real-time PCR, MTB and NTM were cultured in 58 and 29, respectively. The positive rate of real-time PCR assay to detect MTB was 71% (22/31) and 92.6% (25/27) in AFB stain-negative and stain-positive specimens. For NTM, the positive rate of real-time PCR was 11.1% (2/18) and 72.7% (8/11) in AFB stain-negative and stain-positive specimens. Among 63 specimens performed using TB-PCR, MTB and NTM were cultured in 46 and 17, respectively. The positive rate of TB-PCR was 61.7% (21/34) and 100% (12/12) in AFB stain-negative and stain-positive specimens. TB-PCR was negative in all NTM-cultured 17 specimens. CONCLUSION: TB/NTM real-time PCR assay is useful to differentiate MTB and NTM in AFB stain-positive respiratory specimens and it is as effective in detecting MTB with TB-PCR.

ApoE genotyping by real-time fluorescence PCR / 临床检验杂志

Objective To establish a new effective method by using real-time polymerase chain reaction (PCR) to detect single nucleotide polymorphism (SNP) typing for rapid identifying apolipoprotein E alleles.Methods To determine alleles of human apolipoprotein E genetic polymorphism at Cys112Arg locus was detected by PCR melting curve analysis with fluorophore SYBR Green I. In order to increase the speciality of SNP assays, high fidelity Taq polymerase was used. The reliability of SNP typing was validated by comparison with the results of direct DNA sequencing.Results Each sample was determined by double tubes, and two melting curves were analysis. As compared the Tm value of samples with the Tm of standard substance, the apoE genotype of samples was determined. The apoE genotype of 30 samples were E3/3 (27/30) and E3/4 (3/30) respectively, which was accordant with the results of PCR-RFLP and DNA sequencing.Conclusion The presented allelic assay was specific, easy to operate and applicable for discrimination of apolipoprotein E genotyping of human blood.