AIMTo provide more molecular evidences for species relationship between Chuanxiong (Ligusticum chuanxiong Hort.) from China and Japanese Chuanxiong (Senkyu in Japanese) (Cnidium officinale Makino).

METHODSTo sequence such two genes as internal transcribed spacer (ITS) from nuclear rDNA and maturase for lysine (matK) in tRNA(lys) (UUU) intron from chloroplast DNA of both Ligusticum chuanxiong and Cnidium officinale using PCR direct sequencing and to analyze the sequence variation of two genes between these two species.

RESULTSThe matK gene sequence of Ligusticum chuanxiong and Cnidium officinale is 1268 bp in length, coding 422 amino acids of maturase protein. ITS gene sequence 699 bp, consisting of 54 bp of 18S rRNA-3', 215 bp of ITS1, 162 bp of 5.8S rRNA, 222 bp of ITS2, 46 bp of 26S rRNA-5'. Multiple sequence alignment shows that the sequence of two genes between dried crude drug and fresh voucher material of Ligusticum chuanxiong and Cnidium officinale, there is 1 variable site (T-->C) in matK (upstream at 595 nt) and ITS (ITS1 at 54 nt) between Ligusticum chuanxiong and Cnidium officinale.

CONCLUSIONBased on homology analysis of two genes plastid matK and nuclear ITS, the origin of Chuanxiong from China and Japan ought to be identical, the scientific name Cnidium officinale of Japanese Chuanxiong should be changed to Ligusticum chuanxiong.

Amino Acid Sequence ; Base Sequence ; China ; Cnidium ; genetics ; DNA, Plant ; analysis ; DNA, Ribosomal Spacer ; genetics ; Endoribonucleases ; genetics ; Japan ; Ligusticum ; genetics ; Molecular Sequence Data ; Nucleotidyltransferases ; genetics ; Phylogeny ; RNA, Ribosomal, 18S ; genetics ; Sequence Analysis ; Sequence Homology ; Terminology as Topic

Abstract: The first-line drug artemisinin is widely used against malaria. Commercially available artemisinin is extracted from plants. However, the lack of sufficient raw material, artemisinin and the cost associated with the drug's manufacture have limited the supply of ACT to most malaria sufferers in the Developing World. As such, it is important to develop a low cost, fine to environment and high-quality method to supply sufficient and reliable quantities of artemisinin in the future. The field of synthetic biology, which utilizes cell factories to manipulate microbial metabolism to enhance the production of artemisinin and its intermediates, has a particularly strong impact by providing new platforms for chemical production. After a brief introduction of the artemisinin biosynthetic pathway, the present review focuses on the introduction of artemisinin biosynthetic genes, such as the genes encoding amorpha-4, 11-diene monooxygenase, NADPH: cytochrome P450 oxidoreductase, artemisinic aldehyde delta 11(13) reductase and aldehyde dehydrogenase. The review also addresses general considerations for potential contributions of synthetic biology to artemisinin production, with an emphasis on factors influencing interest compounds production in chassis cells.
Antimalarials ; metabolism ; supply & distribution ; Artemisinins ; metabolism ; supply & distribution ; Biosynthetic Pathways ; Cytochrome P-450 Enzyme System ; genetics ; Escherichia coli ; metabolism ; Gene Dosage ; Genetic Engineering ; Isoenzymes ; genetics ; RNA Nucleotidyltransferases ; genetics ; Retinal Dehydrogenase ; genetics ; Saccharomyces cerevisiae ; metabolism ; Synthetic Biology

PURPOSE: The control of tuberculosis is seriously threatened worldwide by the recently emerging multidrug-resistant Mycobacterium tuberculosis. As a result, early detection of drug resistant M.tuberculosis strain has become very important but conventional laboratory methods are time consuming and delayed results often affect patients adversely in controlling tuberculosis. The authors studied the usefulness of the line probe assay to determine the mutaion in rpoB gene of rifampin resistant M.tuberculosis and to find out if this method can substitute conventional methods in the detection of resistant strain. METHODS: This study employed 40 clinical samples of M.tuberculosis which had been determined by culture and drug sensitivity test. After amplification of rpoB-the gene for the B subunit of the RNA polymerase-by PCR, the amplified products were hybridized with specific oligonucleotide probes immobilized on nitrocellulose strip and direct DNA sequencing was also performed. The results were compared with those of the classical susceptibility test. RESULTS: Among the 40 samples, 10 were identified as drug resistant strain by classical drug susceptibility test. Three of the ten resistant samples were rifampin resistant strains, which were identified by either method. All mutations were clustered within the region of 69bp of rpoB and all were single nucleotide mutations. Two isolates had a TCG->TTG(serine->leucine) mutation in codon 522. One isolate had a CAC->CTC(histidine->leucine) mutation in codon 526. CONCLUSION: In contrast to culture and sensitivity tests, line probe assay is an easy and speedy method for detecting rifampin resistant M.tuberculosis in clinical samples as well as a helpful tool for choosing antituberculosis drug in children.
Child ; Codon ; Collodion ; DNA-Directed RNA Polymerases* ; Early Diagnosis* ; Humans ; Mycobacterium tuberculosis* ; Mycobacterium* ; Oligonucleotide Probes ; Polymerase Chain Reaction ; Rifampin ; RNA Polymerase II* ; RNA* ; Sequence Analysis, DNA ; Tuberculosis

OBJECTIVETo investigate gene expression of three nitric oxide synthase isozymes in injured spinal cord tissue.

METHODSThirty-six adult SD rats were randomly divided into six groups: a normal group and five injury groups, with six per each group. Animals in the injury groups were sacrificed at 2, 6, 12, 24, 48 h after injury. A compression injury model on the spinal cord was made according to Nystrom B et al and gene expression of the three NOS isozymes were examined by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSGene expression of nNOS and eNOS were detectable in the normal group and were up-regulated quickly after injury, reaching a maximum at 6 h: (0.633 +/- 0.012) and (1.236 +/- 0.207). Gene expression of iNOS was detectable only in the injury groups and it was gradually up-regulated after injury, reaching a maximum at 24 h: (1.043 +/- 0.049).

CONCLUSIONInjury to the spinal cord leads to early up-regulation of cNOS and late up-regulation of iNOS. Different NOS isozymes may play different roles in secondary spinal cord injury.

Animals ; Female ; Gene Expression Regulation, Enzymologic ; Male ; Nitric Oxide Synthase ; genetics ; Nitric Oxide Synthase Type I ; Nitric Oxide Synthase Type II ; Nitric Oxide Synthase Type III ; RNA ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Spinal Cord ; enzymology ; pathology ; Spinal Cord Injuries ; enzymology ; genetics

OBJECTIVETo investigate the gene and protein expressions of three isoforms of nitric oxide synthase (NOS) and gene expression of Caspase-3, and effect of dexamethasone on them in neonatal rats with lipopolysaccharide (LPS)-induced endotoxemic brain damage.

METHODSExpressions of the three isoforms of NOS and caspase-3 mRNA in the brain were investigated by RT-PCR in postnatal 7-day Wistar rats with acute endotoxemia by intraperitoneal administration of LPS. Regional distributions of NOSs were examined by immunohistochemical technique.

RESULTSnNOS and Caspase-3 mRNA were obviously detected. eNOS mRNA was faintly expressed, but iNOS mRNA was undetectable in the control rat brain. The expressions of NOS mRNA of three isoforms were weak 2 h after LPS (5 mg/mg) delivery, peaked at 6 h, and thereafter, reduced gradually up to 24 h. The expression intensity was in the order of nNOS> iNOS> eNOS. Widespread nNOS, scattered eNOS distribution and negative iNOS were identified in the control rat brain and all isoforms of NOS could be induced by LPS which reached the apex at 24 h in the order of nNOS> iNOS> eNOS as detected by immunostaining. Although Caspase-3 mRNA could be found in all groups, DNA fragmentation was only seen at 6 h and 24 h. The expressions of NOS and Caspase-3 mRNA were inhibited in the rat brain when dexamethasone was administrated.

CONCLUSIONLPS-induced NO production induces apoptosis of neurons through mechanism involving the Caspase-3 activation, which may play an important role in the pathogenesis of brain damage during endotoxemia, and neuro-protective effects of dexamethasone may be partially realized by inhibiting the expression of NOS mRNA.

Animals ; Animals, Newborn ; Apoptosis ; Brain ; drug effects ; enzymology ; Caspase 3 ; Caspases ; genetics ; metabolism ; Dexamethasone ; pharmacology ; Disease Models, Animal ; Endotoxemia ; chemically induced ; enzymology ; Female ; Lipopolysaccharides ; Male ; Nerve Tissue Proteins ; genetics ; metabolism ; Nitric Oxide Synthase ; genetics ; metabolism ; Nitric Oxide Synthase Type I ; Nitric Oxide Synthase Type II ; Nitric Oxide Synthase Type III ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction

The C-terminal domain of RNA polymerase II is an unusual series of repeated residues appended to the C-terminus of the largest subunit and serves as a flexible binding scaffold for numerous nuclear factors. The binding of these factors is determined by the phosphorylation patterns on the repeats in the domain. In this study, we generated a synthetic antibody library by replacing the third heavy chain complementarity-determining region of an anti-HER2 (human epidermal growth factor receptor 2) antibody (trastuzumab) with artificial sequences of 7–18 amino-acid residues. From this library, antibodies were selected that were specific to serine phosphopeptides that represent typical phosphorylation patterns on the functional unit (YSPTSPS)₂ of the RNA polymerase II C-terminal domain (CTD). Antibody clones pCTD-1stS2 and pCTD-2ndS2 showed specificity for peptides with phosphoserine at the second residues of the first or second heptamer repeat, respectively. Additional clones specifically reacted to peptides with phosphoserine at the fifth serine of the first repeat (pCTD-1stS5), the seventh residue of the first repeat and fifth residue of the second repeat (pCTD-S7S5) or the seventh residue of either the first or second repeat (pCTD-S7). All of these antibody clones successfully reacted to RNA polymerase II in immunoblot analysis. Interestingly, pCTD-2ndS2 precipitated predominately RNA polymerase II from the exonic regions of genes in genome-wide chromatin immunoprecipitation sequencing analysis, which suggests that the phosphoserine at the second residue of the second repeat of the functional unit (YSPTSPS)2 is a mediator of exon definition.
Antibodies ; Chromatin Immunoprecipitation ; Clone Cells ; Complementarity Determining Regions ; DNA-Directed RNA Polymerases* ; Exons* ; Peptides ; Phosphopeptides ; Phosphorylation* ; Phosphoserine ; Receptor, Epidermal Growth Factor ; RNA Polymerase II* ; RNA* ; Sensitivity and Specificity ; Serine

BACKGROUND: The emergence of multidrug-resistant strains of Mycobacterium tuberculosis presents a significant challange to the treatment and control of tuberculosis, and there is an urgent need to understand the mechanisms by which strains acquire multidrug resistance. Recent advances in molecular methods for the detection of M. tuberculosis genetic targets have approached the sensitivity of culture Furthermore the prospect of determining resistance in mycobacteria at the nucleic acid level particulary to first-line drugs like rifampin, isoniazid has provided a glimps of the next generation of sensitivity test for M. tuberculosis. Previous studies in RMP resistant M. tuberculosis have shown that mutation in beta subunit of RNA polymerase is main mechanism of resistance. METHOD: In this study, rpoB gene for the ~3 subunit of RNA polymerase from M. tuberculosis of 42 cultured samples (32 were RMP resistant and 10 were sensitive cases) were isolated and characterised the mutations. Direct sequencing data were compared with the results of INNO-LiPA Line Probe Assay (LiPA, Innogenetics, Belgium), commercial RMP resistance detecting kit using reverse hybridization method. RESULTS: All of the RMP resistant samples were revealed the presence of mutation by LiPA. In 22 samples (68.8%) out of 32 RMP resistant cases, the mutation types were confirmed by the positive signal at one of 4 mutation bands in the strip. The most frequent type was R5 (S53 IL) which were 17 cases (77.3%). Results of direct sequencing were identified the exact characteristics of 8 mutations which were not comfirmed by LiPA. S522W type point mutation and 9 base pair deletion at codon 513-515 were new identified mutations for the first time. CONCLUSION: Mutations in rpoB gene is the main mechanism of RMP resistance in M. tuberculosis and LiPA is a very useful diagnostic tool for the early diagnosis of RMP resistance in M. tuberculosis.
Base Pairing ; Codon ; DNA-Directed RNA Polymerases ; Drug Resistance, Multiple ; Early Diagnosis ; Isoniazid ; Mycobacterium tuberculosis ; Point Mutation ; Rifampin ; RNA Polymerase I ; Tuberculosis*

BACKGROUNDThe role of epigenetics in gene expression regulation and development significantly enhances our understanding of carcinogenesis. All the tumor related genes may be the target of epigenetical or genetic regulation. We selected some epigenetically regulated genes for cDNA array analysis and observed variability in the expression of the DICER1 gene in distinct stages of gastric cancer. The aim of this study was to assess the correlation between the expression of DICER1, an epigenetically regulated gene, and gastric cancer.

METHODSTo detect the expression of 506 tumor-associated genes, including DICER1, in the matched cancerous mucosa, pre-malignant lesion (adjacent mucosa), non-cancerous gastric mucosa and distant lymphocyte metastatic lesion in 3 cases of gastric cancers using cDNA array. DICER1 mRNA expression and DICER1 protein expression were further analyzed by Real-time PCR and Western blot in 32 cases of progressive gastric cancer. DICER1 protein expression was also detected in 33 early and 30 progressive gastric cancers by the immunohistochemistry (IHC) method.

RESULTSIn 3 cases of gastric cancer cDNA array showed dramatically decreased expression of DICER1 in pre-malignant lesion, cancerous mucosa and distant lymphocyte metastatic lesions compared with matched noncancerous gastric mucosa, pre-malignant lesion and cancerous mucosa. Real-time PCR results showed that the expression level of DICER1 mRNA in gastric cancer was significantly down-regulated compared to normal gastric tissue (P < 0.05). The IHC assay also showed that the expression of DICER1 was significantly decreased in progressive gastric cancer. Among the 63 cases of gastric cancers, 13/33 early (39.4%) and 19/30 (63.3%) progressive cancers showed negative expression of DICER1 (50.8%). The difference in expression of DICER1 between early and progressive gastric cancers was significant (P < 0.01). The result of Western blotting showed that DICER1 protein was down-regulated significantly in advanced gastric cancer (P < 0.05).

CONCLUSIONSDICER1 expression is decreased during the progression of gastric cancer, especially in progressive gastric cancers, which indicating DICER1 may play an important role in the development of cancer and the epigenetical regulation involved.

Blotting, Western ; DEAD-box RNA Helicases ; analysis ; genetics ; physiology ; Endoribonucleases ; analysis ; genetics ; physiology ; Epigenesis, Genetic ; Humans ; Immunohistochemistry ; Oligonucleotide Array Sequence Analysis ; Polymerase Chain Reaction ; Ribonuclease III ; Stomach Neoplasms ; chemistry ; etiology ; genetics

OBJECTIVETo analyze the association of micoRNA-related genes DROSHA single nucleotide polymorphisms (SNP) rs10719 and rs6877842, DICER1 rs3742330and GEMIN4 rs3744741 with prognosis of T-cell lymphoma.

METHODSPolymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to determine the genotypes of the above 4SNPs and their associations with complete remission (CR) rate and overall survival (OS) in 163 patients with TCL.

RESULTSPatients carrying the rs6877842 CG genotype had a significantly higher CR rate compared with those carrying the CC genotype (OR=0.07, 95% CI 0.01-0.72, P=0.026); the same for patients carrying the DICER1 rs3742330 GG genotype compared with those carrying the GA genotype (OR=0.15, 95% CI 0.02-0.97, P=0.047) or the AA genotype (OR=0.11, 95% CI 0.02-0.71, P=0.020). In addition, patients with the DICER1 rs3742330 GG genotype had a significantly improved OS compared with those carrying the GA (HR=9.02, 95% CI 1.22-66.92, P=0.031) or AA genotype (HR=8.77, 95% CI 1.19-64.67, P=0.033). The other two SNPs of rs10719 and rs3744741 had no significant association with CR or OS.

CONCLUSIONDROSHA rs6877842 and DICER1 rs3742330 were independent factors for TCL CR, and DICER1 rs3742330 was also an independent prognostic factor for TCL OS.

DEAD-box RNA Helicases ; genetics ; Genetic Predisposition to Disease ; Genotype ; Humans ; Lymphoma, T-Cell ; diagnosis ; genetics ; MicroRNAs ; genetics ; Minor Histocompatibility Antigens ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; Prognosis ; Ribonuclease III ; genetics ; Ribonucleoproteins, Small Nuclear ; genetics

The swine influenza virus (SIV) strain A/Swine/TianJin/01/2004(H1N1) (A/S/TJ/04) was rescued successfully by an eight-plasmid rescue system. The cDNAs of SIV 8 gene segments were synthesized by RT-PCR and cloned into the RNA polymerase I/II bidirection expression vector PHW2000 independently, resulting in 8 recombinant plasmids. The 8 recombinant plasmids were cotransfected into COS-1 cell, 30 h later TPCK-trypsin was added to 0.5 microg/mL. The COS-1 cell and supernatant were harvested 48 h after cotransfection and were inoculated into the allantoic cavity of 9-day-old specific-pathogen free (SPF) chicken eggs. The allantoic fluid of dead eggs was harvested and passaged 3 generations in SPF chicken eggs to get infective virus. The successful rescue of A/S/TJ/04 SIV was identified by hemagglutination assay, hemagglutination inhibition assay, sequence analysis and electron microscope observation. The successful rescue of SIV built a platform for the research of the relationship between genome structure and function of SIV, the mechanisms of trans-species transmission of influenza virus and for the generation of new SIV as vaccine.
Animals ; COS Cells ; Cercopithecus aethiops ; Chick Embryo ; Chickens ; Influenza A Virus, H1N1 Subtype ; genetics ; physiology ; Plasmids ; genetics ; RNA Polymerase I ; genetics ; RNA Polymerase II ; genetics ; Recombination, Genetic ; genetics ; Swine ; Transfection ; Virus Replication