This study is to investigate the effect of artesunate on transforming growth factor-beta1 (TGF-beta1) induced epithelial-mesenchymal transition (EMT) and its possible mechanism. After the in vitro cultured RLE-6TN cells were treated with TGF-beta1 then artesunate intervened on it, after 24 h, expression of the markers of mesenchymal cell was assayed using Western blotting and real-time PCR analysis. Western blotting was also used to detect the effect of TGF-beta1 on the Smad3 and Smad7 expressions of RLE-6TN cells. Morphological alterations were examined by phase-contrast microscope, and ultrastructure changes by electron microscope. Incubation of RLE-6TN cells with TGF-beta1 resulted in the up-regulation of the expression of the mesenchymal cell markers, after artesunate intervened on it, resulted in the down-regulation of the expression. Meanwhile, incubation with artesunate intervened on RLE-6TN cells could lead to the apparent down-regulation of the expression of Smad3 and up-regulation of Samd7 and the transition of RLE-6TN cells to mesenchymal-like by TGF-beta1 induction, after artesunate intervened on it, RLE-6TN cells to epithelial-like. TGF-beta1 induced epithelial-mesenchymal transition process; artesunate can inhibit TGF-beta1-induced epithelial-mesenchymal transition process, the possible mechanism is up-regulation of the expression of Smad7 and down-regulation of the expression of Smad3, meanwhile inhibits phosphorylation of Smad3.
Actins ; genetics ; metabolism ; Animals ; Artemisia ; chemistry ; Artemisinins ; isolation & purification ; pharmacology ; Cell Line ; Cell Proliferation ; drug effects ; Epithelial Cells ; cytology ; metabolism ; Epithelial-Mesenchymal Transition ; drug effects ; Idiopathic Pulmonary Fibrosis ; pathology ; Plants, Medicinal ; chemistry ; Pulmonary Alveoli ; cytology ; RNA, Messenger ; metabolism ; Rats ; Smad3 Protein ; genetics ; metabolism ; Smad7 Protein ; genetics ; metabolism ; Transforming Growth Factor beta1 ; pharmacology ; Vimentin ; genetics ; metabolism

According to the existing Provisions for Drug Registration (SFDA Order No. 28), applications for new drugs of traditional Chinese medicine are divided into two parts: the applications for drug clinical trial and for drug production (including new drug certificate). It will last for about 10 years from the application for drug clinical trial to get approving, and it also remains many problems and the low probability to succeed. From the sight of pharmaceutical review, there are mainly two aspects of regulatory compliance and technical issues, mainly for changes without approval of the competent authorities of the country. For example, sample preparation and approval of clinical trial process are significant changes. Technical problems are reporting incomplete data or information submitted does not comply with the technical requirements for review, such as: production process validation does not provide information, the preparation of samples for clinical trials and field inspection, production information, or the information provided does not meet the technical requirements. This paper summarizes the frequently asked questions and to make recommendations to advise applicants concerned, timely detection of problems, avoid risk, improving the quality and efficiency of the application for registration.
China ; Drug Approval ; legislation & jurisprudence ; Drug Evaluation ; legislation & jurisprudence ; Humans ; Legislation, Drug ; Medicine, Chinese Traditional

China ; History, 20th Century ; History, 21st Century ; Pathology ; Periodicals as Topic ; history

The nitrite-reducing activity of aerobic denitrifying bacterial strain N6-1 was studied. It showed that the nitrite-reducing activity reached the highest at 30℃, 120 r/min, pH 8.5 and C/N ratio 12, using CH3COONa and NaNO2 as the sole carbon source and nitrogen source, respectively. When the initial NaNO2 concentration was 2 g/L, NO2--N was reduced completely after 20 hours cultivation with the reducing rate of 20.3 mg/L?h. There would be no effect on its nitrite-reducing activity in the present of 1.5% NaCl or 1% peptone. The cell concentration could reach 1.2?1011 CFU/mL after 24 hours cultivation in 10 L fermentor.

S1 nuclease (from Aspergillus oryzae) is a specific enzyme to degrade single stranded DNA or RNA molecules. It has been reported to be able to convert superhelical circular DNA molecules into open circle or linear forms under certain conditions, but this function has not been well explored. In order to use the action of S1 nuclease to linearize circular DNA and develop a novel way of cloning microcircular DNAs, the pUC19 was used to investigate the relationship between the linearization efficiency of S1 nuclease and the amount of enzyme used. By this way the optimal conditions for linearization of circular DNAs by S1 nuclease would be determined. 0.3u to 17u S1 nuclease per 100ng pUC19 DNA was added into a 25 microL system, respectively, to perform the reaction. The effectiveness of enzyme digestion was realized by electrophoresis in a 1.2% agarose gel. The results showed that along with the increase in enzyme amount from 0.3u to 17u a gradual decrease in the superhelical form, a gradual increase in the linear form and then in the circular form was obvious. The conversion from superhelical form to linear and circular form was directly related to the enzyme amount used. A higher proportion of linear DNA molecules was achieved by using 5 to 17u S1 nuclease per 100ng DNA. Besides, electrophoretic mobility of the S1 nuclease-linearized pUC19 was the same as that of the linear form produced by restriction enzyme digestion. According to the result of phiX174 digested by S1 nuclease it has been proposed that the enzyme cleaves first randomly on one site of one strand, thus converting the superhelical molecules into open circle form, and then on the same site of the complementary strand to produce the linear form. Therefore, the S1 nuclease-linearized DNA molecules are intact in the sense of their length and can be used for cloning. The plasmid-like DNA pC3 from cucumber mitochondria is a double stranded circular DNA molecule with about 550bp and the smallest known plasmid-like DNA in eukaryotic mitochondria. Many attempts have been made to linearize the molecule by using restriction enzymes but failed. Therefore, S1 nuclease was used to linearize pC3 based on the results obtained with pUC19. The linearized pC3 DNA molecules formed a very sharp band in a 2.5% agarose gel after electrophoresis. They were then recovered from the gel, added an "A" tail and ligated with T-vector. After transformation into E. coli JM109 cells, the positive clones were, screened by the blue-white selection. The insert was then cut using restriction enzymes EcoRI and Pst I. The result of electrophoresis shows that the electrophoretic mobility of the insert is just the same as that predicted. A 32 P-labled probe was synthesized using pC3 as the template and Southern blot analysis was carried out. The result shows that the inserted DNA is hybridized to the probe, which indicates that the cloned DNA fragment is from pC3. The sequence information of the insert shows that the plasmid-like DNA pC3 was 537bp in length. The nucleotide sequence was deposited in the GenBank (the accession number is AF522195).
Blotting, Southern ; Cloning, Molecular ; methods ; DNA, Circular ; genetics ; metabolism ; Fungal Proteins ; genetics ; metabolism ; Molecular Sequence Data ; Single-Strand Specific DNA and RNA Endonucleases ; genetics ; metabolism

OBJECTIVETo screen and identify differentially expressed genes in the hippocampus of the offsprings of lead exposed female rats in order to provide a theoretical basis for identifying learning and memory deficits related genes.

METHODSRNA was extracted from the hippocampus of young rats with learning and memory deficits due to maternal lead exposure. Suppression subtractive hybridization was used to identify the differentially expressed genes in the hippocampus.

RESULTSAn effective subtracted library was constructed which consisted of approximately 200 clones. Sequencing for the library identified 93 clones harboring insertion fragments which included 43 different genes and 4 unknown genes. These genes might be related to learning and memory deficits due to maternal lead exposure.

CONCLUSIONSThe up-regulated genes in the hippocampus of young rats from pregnant rats under lead exposure include some housekeeping genes and some proteins involved in cellular protein folding, signal transduction, stress response and DNA methylation. These proteins might be directly related to a significant reduction in learning and memory abilities in the young rats.

Animals ; Female ; Gene Expression Profiling ; Gene Library ; Hippocampus ; drug effects ; metabolism ; Lead ; toxicity ; Learning ; drug effects ; Memory ; drug effects ; Nucleic Acid Hybridization ; methods ; Polymerase Chain Reaction ; Pregnancy ; Rats

Adult ; Diagnosis, Differential ; Female ; Humans ; Ovarian Neoplasms ; pathology ; Sertoli-Leydig Cell Tumor ; pathology

The applications accepted and approved by general program, young scientist fund and fund for less developed region of national natural science funds in the discipline of Chinese materia medica, NSFC in 2012 have been introduced. The research contents of the funded projects in the popular research areas have been summarized and the problems in the applications have been analyzed to give a reference to the scientists in the field of Chinese materia medica.
China ; Financing, Organized ; organization & administration ; Humans ; Laboratory Personnel ; economics ; Materia Medica ; chemistry ; Medicine, Chinese Traditional ; economics ; Natural Science Disciplines ; economics ; manpower ; organization & administration

Adult ; Carcinoma ; virology ; Cervical Intraepithelial Neoplasia ; virology ; Female ; Human papillomavirus 16 ; isolation & purification ; Human papillomavirus 18 ; isolation & purification ; Humans ; In Situ Hybridization ; Middle Aged ; Papillomavirus Infections ; Uterine Cervical Neoplasms ; virology

OBJECTIVETo investigate the effects of microRNA (miRNA) on proliferation of cultured human squamous cell carcinoma of tongue Tca8113 cells.

METHODSThe mimics or inhibitors of miRNA-31 or miRNA-139 were transfected into Tca8113 cells using liposome. Tca8113 cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.

RESULTSThe absorbance (A) values of control group at 24 h, 48 h and 72 h were 0.125 +/- 0.002, 0.169 +/- 0.002 and 0.216 +/- 0.004, respectively. The mimics of miRNA-31 increased Tca8113 cell proliferation, with A values increasing to 0.136 +/- 0.001 (P < 0.001), 0.186 +/- 0.004 (P < 0.001) and 0.249 +/- 0.012 (P < 0.01), respectively. The inhibitors of miRNA-139 also increased A values to 0.148 +/- 0.002 (P < 0.001), 0.214 +/- 0.002 (P < 0.001) and 0.250 +/- 0.009 (P < 0.01), respectively. Contrast with these results, the inhibitors of miRNA-31 decreased Tca8113 cell proliferation, with A values decreasing to 0.145 +/- 0.001 and 0.155 +/- 0.011 (both of P < 0.001) at 48 h and 72 h, respectively. The mimics of miRNA-139 also decreased A to 0.135 +/- 0.001 and 0.170 +/- 0.009 (both of P < 0.001).

CONCLUSIONSmiRNA-31 and miRNA-139 play an important role in the carcinogenesis of human tongue carcinomas. It may become a new method for the treatment of tongue carcinomas by adjustment the activities of miRNA.

Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Humans ; MicroRNAs ; antagonists & inhibitors ; metabolism ; Tongue Neoplasms ; genetics ; metabolism ; pathology ; Transfection