"Wu zhu yu", which is obtained from the dried unripe fruits of Tetradium ruticarpum (A. Jussieu) T. G. Hartley, has been used as a traditional Chinese medicine for treatment of headaches, abdominal colic, and hypertension for thousands of years. The present study was designed to assess the molecular genetic diversity among 25 collected accessions of T. ruticarpum (Wu zhu yu in Chinese) from different areas of China, based on inter-primer binding site (iPBS) markers and inter-simple sequence repeat (ISSR) markers. Thirteen ISSR primers generated 151 amplification bands, of which 130 were polymorphic. Out of 165 bands that were amplified using 10 iPBS primers, 152 were polymorphic. The iPBS markers displayed a higher proportion of polymorphic loci (PPL = 92.5%) than the ISSR markers (PPL = 84.9%). The results showed that T. ruticarpum possessed high loci polymorphism and genetic differentiation occurred in this plant. The combined data of iPBS and ISSR markers scored on 25 accessions produced five clusters that approximately matched the geographic distribution of the species. The results indicated that both iPBS and ISSR markers were reliable and effective tools for analyzing the genetic diversity in T. ruticarpum.
Base Sequence ; Binding Sites ; DNA Fingerprinting ; DNA Primers ; metabolism ; DNA, Plant ; genetics ; isolation & purification ; Evodia ; classification ; genetics ; Genetic Markers ; genetics ; Genetic Variation ; Interspersed Repetitive Sequences ; genetics ; Phylogeny ; Polymorphism, Genetic ; Random Amplified Polymorphic DNA Technique ; Terminal Repeat Sequences ; genetics

OBJECTIVETo explore the molecular basis for a CD36 deficiency individual and distribution of CD36 gene mutation in Guangxi population.

METHODSA female individual was studied. CD36 phenotype was detected by monoclonal antibody immobilization of platelet antigens assay (MAIPA) and flow cytometry (FCM). The coding regions of the CD36 gene were sequenced. A DNA-based polymerase chain reaction-sequence specific primer (PCR-SSP) assay was used to verify the identified mutation. Cell lines expressing the mutant and wild-type CD36[CD36(MT) and CD36(WT)] were established, with the expression of CD36 determined by Western blotting. The distribution of CD36 gene mutation was investigated among 1010 unrelated individuals with the PCR-SSP assay.

RESULTSBoth MAIPA and FCM assays showed that the patient had type II CD36 deficiency. DNA sequencing showed that she has carried a heterozygous mutation T538C (Trp180Arg) in the exon 6 of CD36. Sequencing of cDNA clone confirmed that there was a nucleotide substitution at position 538 (538T>C). Western blotting also confirmed that the CD36 did not express on the CD36(MT) cell line that expressed the 538C mutant, but did express on the CD36(WT) cell line. The novel CD36 mutation T538C was further verified with 100% concordance of genotyping results by DNA-based PCR-SSP assay and 1010 unrelated individuals. No CD36 538C allele was detected among the 1010 individuals.

CONCLUSIONThis study has identified a novel CD36 mutation T538C(Trp180Arg)(GenBank: HM217022.1), and established a genotyping method for the novel sequence-specific primer PCR. The novel mutation is rare in Guangxi and can cause type II CD36 deficiency.

Base Sequence ; Blood Platelet Disorders ; genetics ; Blood Platelets ; cytology ; metabolism ; Blotting, Western ; CD36 Antigens ; genetics ; metabolism ; Cells, Cultured ; DNA Mutational Analysis ; DNA Primers ; genetics ; Exons ; genetics ; Female ; Flow Cytometry ; Fluorescent Antibody Technique ; Genetic Diseases, Inborn ; genetics ; Genotype ; Genotyping Techniques ; methods ; Humans ; Middle Aged ; Monocytes ; cytology ; metabolism ; Mutation, Missense ; Polymerase Chain Reaction ; methods

BACKGROUND: Several molecular assays have been developed to detect the BRAF V600E mutation in fine needle aspirates (FNAs) for the diagnosis of papillary thyroid cancer. Using a multiplex PCR technique, we evaluated the Anyplex BRAF V600E Real-time Detection (Anyplex) assay and compared its efficacy with that of the Seeplex BRAF V600E ACE Detection (Seeplex) method. METHODS: We tested 258 consecutive FNA specimens using the Seeplex and Anyplex assays. Any conflicting results between the two assays were confirmed by using mutant enrichment with 3'-modified oligonucleotide (MEMO) sequencing. The limits of detection (LODs) and reproducibility for each assay were evaluated with serially diluted DNA from a BRAF V600E-positive cell line. RESULTS: The BRAF V600E mutation was detected in 36.4% (94/258) FNA specimens by either the Seeplex or Anyplex assay. Results for the two assays showed 93.4% (241/258) agreement, with a kappa value of 0.861 (95% confidence interval, 0.798-0.923). Of the eight specimens that were BRAF V600E-positive by the Anyplex assay but not by the Seeplex assay, five were found to be BRAF V600E-positive by MEMO sequencing. The mutation detection rate of the Seeplex and Anyplex assays was 79.0% and 84.0%, respectively, in the FNA specimens diagnosed as malignant (n=81). The LOD as determined by probit analysis was 0.046% (95% confidence interval, 0.019-0.532%). CONCLUSIONS: The Anyplex assay performed better than the Seeplex assay with respect to the detection of the BRAF V600E mutation.
Adult ; Aged ; Asian Continental Ancestry Group/genetics ; Biopsy, Fine-Needle ; DNA/chemistry/metabolism ; DNA Mutational Analysis/*methods ; DNA Primers/*metabolism ; Female ; Humans ; Male ; Middle Aged ; Multiplex Polymerase Chain Reaction ; Oligonucleotides/metabolism ; Polymorphism, Single Nucleotide ; Proto-Oncogene Proteins B-raf/*genetics ; Republic of Korea ; Thyroid Nodule/*metabolism/pathology

miR-146a is an immunoregulatory microRNA closely associated with viral infection. This study investigated the expression changes of miR-146a in peripheral blood monocytes of HCV-infected patients and the mechanism by which the THP-1 cells were stimulated with HCV core protein in vitro. It was found that in the peripheral blood monocytes of HCV-infected patients, miR-146a expression was upregulated. After treated by interferon/ribavirin, miR-146a expression was decreased when HCV RNA became undetectable. HCV core could directly stimulate THP-1 cells to produce miR-146a. Silencing TLR2 and MyD88 could significantly inhibit the expression of miR-146a. It was concluded that the expression of miR-146a in peripheral blood monocytes of HCV-infected patients was abnormally increased. The TLR2-MyD88 signaling pathway may take part in the overexpression of miR-146a in monocytes stimulated with HCV core protein.
Adult ; Base Sequence ; Cell Line ; DNA Primers ; Female ; Hepatitis C, Chronic ; blood ; Humans ; Male ; MicroRNAs ; blood ; Middle Aged ; Monocytes ; metabolism ; Myeloid Differentiation Factor 88 ; physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Toll-Like Receptor 2 ; physiology ; Young Adult

This study investigated the expression of lung surfactant proteins SP-B and SP-C, and their modulating factors TTF-1 and PLAGL2 in the fetal lung of rats with fetal growth restriction (FGR). The rat FGR model was established by prenatal hypoxia in the first stage of pregnancy, 180 rats for experiment served as hypoxia group, and 197 healthy rats served as normal control group. The FGR incidence in hypoxia was compared with that in normal control group. The histological changes in the fetal lung were observed under the light microscope and electronic microscope in two groups. The SP-B, SP-C, TTF-1 and PLAGL2 proteins were determined in the fetal lung of two groups immunohistochemically. The expression levels of SP-B, SP-C, TTF-1 and PLAGL2 protein and mRNA in the fetal lung of two groups were detected by using Western blotting and RT-PCR respectively. The FGR rat model was successfully established by using hypoxia. Pathologically the fetal lung developed slowly, and the expression levels of SP-B, SP-C, TTF-1 and PLAGL2 protein and mRNA in the fetal lung were significantly reduced in hypoxia group as compared with those in normal control group. It was suggested that maternal hypoxia in the first stage of pregnancy could induce FGR, and reduce the expression of SP-B and SP-C, resulting in the disorder of fetal lung development and maturation.
Animals ; Base Sequence ; DNA Primers ; Female ; Fetal Growth Retardation ; Lung ; embryology ; metabolism ; Peptides ; metabolism ; Pregnancy ; Pulmonary Surfactant-Associated Protein B ; metabolism ; Rats ; Real-Time Polymerase Chain Reaction

Systemic lupus erythematosus (SLE) and clear cell renal cell carcinoma (CC-RCC) are serious disorders and usually fatal, and always accompanied with pathological changes in the kidney. Signal-induced proliferation-associated protein 1 (SIPA-1) is a Rap1GTPase activating protein (Rap1GAP) expressed in the normal distal and collecting tubules of the murine kidney. Lupus-like autoimmune disease and leukemia have been observed in SIPA-1 deficient mice, suggesting a pathological relevance of SIPA-1 to SLE and carcinoma in human being. The expression pattern of SIPA-1 is as yet undefined and the pathogenesis of these diseases in humans remains elusive. In this study, we used both immunohistochemistry and quantum dot (QD)-based immunofluorescence staining to investigate the expression of SIPA-1 in renal specimens from SLE and CC-RCC patients. MTT assay and Western blotting were employed to evaluate the effects of SIPA-1 overexpression on the proliferation and apoptosis of renal cell lines. Semi-quantitative reverse transcriptase-PCR (RT-PCR) was applied to examine the changes of hypoxia-inducible factor-1α (HIF-1α) mRNA level. Results showed that SIPA-1 was highly expressed in the proximal and collecting tubules of nephrons in SLE patients compared to normal ones, and similar results were obtained in the specimens of CC-RCC patients. Although SIPA-1 overexpression did not affect cellular proliferation and apoptosis of both human 786-O renal cell carcinoma cells and rat NRK-52E renal epithelial cell lines, RT-PCR results showed that HIF-1α mRNA level was down-regulated by SIPA-1 overexpression in 786-O cells. These findings suggest that SIPA-1 may play critical roles in the pathological changes in kidney, and might provide a new biomarker to aid in the diagnosis of SLE and CC-RCC.
Apoptosis ; Base Sequence ; Cell Line ; Cell Proliferation ; DNA Primers ; GTPase-Activating Proteins ; metabolism ; Humans ; Kidney Tubules, Proximal ; metabolism ; pathology ; Lupus Erythematosus, Systemic ; metabolism ; pathology ; Nuclear Proteins ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

The aim of this study was to investigate the possible beneficial effects of Fenofibrate on renal ischemia-reperfusion injury (IRI) in mice and its potential mechanism. IRI was induced by bilateral renal ischemia for 60 min followed by reperfusion for 24 h. Eighteen male C57BL/6 mice were randomly divided into three groups: sham-operated group (sham), IRI+saline group (IRI group), IRI+Fenofibrate (FEN) group. Normal saline or Fenofibrate (3 mg/kg) was intravenously injected 60 min before renal ischemia in IRI group and FEN group, respectively. Blood samples and renal tissues were collected at the end of reperfusion. The renal function, histopathologic changes, and the expression levels of pro-inflammatory cytokines [interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-α) and IL-6] in serum and renal tissue homogenate were assessed. Moreover, the effects of Fenofibrate on activating phosphoinositide 3 kinase/protein kinase B (PI3K/Akt) signaling and peroxisome proliferator-activated receptor-α (PPAR-α) were also measured in renal IRI. The results showed that plasma levels of blood urea nitrogen and creatinine, histopathologic scores and the expression levels of TNF-α, IL-8 and IL-6 were significantly lower in FEN group than in IRI group. Moreover, Fenofibrate pretreatment could further induce PI3K/Akt signal pathway and PPAR-α activation following renal IRI. These findings indicated PPAR-α activation by Fenofibrate exerts protective effects on renal IRI in mice by suppressing inflammation via PI3K/Akt activation. Thus, Fenofibrate could be a novel therapeutic alternative in renal IRI.
Animals ; Base Sequence ; DNA Primers ; Enzyme Activation ; Fenofibrate ; pharmacology ; therapeutic use ; Inflammation ; drug therapy ; Kidney ; blood supply ; Male ; Mice ; Mice, Inbred C57BL ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Real-Time Polymerase Chain Reaction ; Reperfusion Injury ; drug therapy ; Signal Transduction

The purpose of this study was to investigate the potential cardioprotection roles of Rapamycin in anoxia/reoxygenation (A/R) injury of cardiomyocytes through inducing autophagy, and the involvement of PI3k/Akt pathway. We employed simulated A/R of neonatal rat ventricular myocytes (NRVM) as an in vitro model of ischemial/reperfusion (I/R) injury to the heart. NRVM were pretreated with four different concentrations of Rapamycin (20, 50, 100, 150 μmol/L), and pretreated with 10 mmol/L 3-methyladenine (3MA) for inhibiting autophagy during A/R. Then, Western blot analysis was used to examine variation in the expression of LC3-II, LC3-I, Bim, caspase-3, p-PI3KI, PI3KI, p-Akt and Akt. In our model, Rapamycin had a preferential action on autophagy, increasing the expression of LC3-II/LC3-I, whereas decreasing the expression of Bim and caspase-3. Moreover, our results also demonstrated that Rapamycin inhibited the activation of p-PI3KI and enhanced the activation of p-Akt. It is concluded that Rapamycin has a cardioprotection effect by inducing autophagy in a concentration-dependent manner against apopotosis through PI3K/Akt signaling pathway during A/R in NRVM.
Animals ; Autophagy ; drug effects ; Base Sequence ; Cells, Cultured ; DNA Primers ; Myocytes, Cardiac ; drug effects ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Real-Time Polymerase Chain Reaction ; Reperfusion Injury ; prevention & control ; Sirolimus ; pharmacology

Intermediate-resolution HLA-DQ typing has gained importance in organ transplantation recently. We evaluated the performance of the LIFECODES HLA-DQB1 typing kit (Immucor, USA) using sequence-specific oligonucleotide (SSO) probe and Luminex platform (Luminex Corp., USA) on 100 samples tested by sequence-based typing (SBT) using the AlleleSEQR HLA-DQB1 kit (Abbott Molecular, USA) in Korean individuals. No sample showed ambiguity in the assignment of 4-digit HLA-DQB1 allele with the LIFECODES HLA-DQB1 SSO typing kit, and the results were fully concordant with those of high-resolution typing of AlleleSEQR HLA-DQB1 SBT up to 4-digit level. Three samples required adjustment of false reactions (3/100, 3.0%): two samples with DQB1*03:03/*06:01 showed false-positive result in probe 253, and 1 sample with DQB1*04:02/*05:02 showed false-negative result in probe 217. We tested an additional sample with DQB1*03:03/*06:01, which showed same false-positivity in probe 253 and 2 samples with DQB1*04:02/*05:02, which showed no false reaction. The false reactions did not result in ambiguity or change in the HLA allele assignment. We could assign HLA-DQB1 alleles to 4 digit-level without ambiguity, with 100% concordance with the SBT results. Thus, LIFECODES HLA-DQB1 SSO typing kit showed good performance for intermediate-resolution HLA-DQB1 typing in clinical laboratory for organ transplantation in Koreans.
Alleles ; DNA Primers/metabolism ; Gene Frequency ; Genotype ; HLA-DQ beta-Chains/*genetics/metabolism ; Histocompatibility Testing/*standards ; Humans ; Polymerase Chain Reaction ; Reagent Kits, Diagnostic/*standards ; Republic of Korea

The cyclization of 2,3-oxidosqualene is the key branch point of ergosterol and triterpenoid biosynthesis. Downregulation of 2,3-oxidosqualene metabolic flux to ergosterol in Saccharomyces cerevisiae may redirect the metabolic flux toward the triterpenoid synthetic pathway. In our study, primers were designed according to erg7 gene sequence of S. cerevisiae. Three fragments including 5' long fragment, 5' short fragment and erg7 coding region fragment were amplified by PCR. 5' long fragment consists of the promoter and a part of erg7 coding region sequence. 5' short fragment consists of a part of promoter and a part of erg7 coding region sequence. These fragments were inserted reversely into pESC-URA to construct antisense expression plasmids. The recombinant plasmids were transformed into S. cerevisiae INVSc1 and recombinant strains were screened on the nutritional deficient medium SD-URA. The erg7 expression level of recombinant strains, which harbored antisense expression plasmid of erg7 coding region, was similar to that of INVScl by semi-quantitative PCR detection. But erg7 expression level of recombinant strains, which harbored 5' long antisense fragment and 5' short antisense fragment, was significantly lower than that of the control. The results of TLC and HPLC showed that the ergosterol content of recombinant strains, which harbored 5' long antisense fragment, decreased obviously. The ergosterol contents of the others were almost equal to that of INVSc1. Lanosterol synthase gene expression was downregulated by antisense RNA technology in S. cerevisiae, which lays a foundation for reconstructing triterpenoid metabolic pathway in S. cerevisiae by synthetic biology technology.
DNA Primers ; Down-Regulation ; Gene Expression ; Intramolecular Transferases ; genetics ; metabolism ; Plasmids ; Polymerase Chain Reaction ; RNA, Antisense ; Saccharomyces cerevisiae ; enzymology ; genetics ; Squalene ; analogs & derivatives ; metabolism ; Transformation, Genetic