OBJECTIVE: To establish two methods by denaturing gradient gel electrophoresis (DGGE) and pyrosequencing for genotyping rs220030 (a SNP in the promoter region of small nuclear ribonucleoprotein polypeptide N, SNRPN). To establish an analytical technique for detecting CpG methylation status by pyrosequencing and to further investigate the feasibility of applying rs220030 to the determination of parental origin allele. METHODS: The rs220030 of 97 blood samples from individuals of Shanghai Han population were genotyped by DGGE, meanwhile the rs220030 of 25 blood samples of them were genotyped by pyrosequencing to compare the two methods in genotyping SNP. Pyrosequencing united bisulfite conversion method was applied to detect CpG methylation status of region upstream rs220030 of two random blood genealogical samples and investigate whether the methylation status was parental related. RESULTS: The rs220030 genotyping results of 97 blood samples detected by DGGE were 20 C homozygote, 29 T homozygote, and 48 C/T heterozygote. Twenty-five blood samples genotyped by pyrosequencing showed the same result with DGGE. The CpG methylation status of region upstream rs220030 of the child was similar to the mother. CONCLUSION Compared with DGGE, pyrosequencing is more accurate, convenient, and suitable for large samples and high throughput SNP genotyping. Pyrosequencing united bisulfite conversion can be used to detect CpG methylation status precisely. It is feasible to apply rs220030 to parental origin allele determination.
Asian People/genetics* ; CpG Islands ; DNA/genetics* ; DNA Methylation ; DNA Primers ; Genomic Imprinting ; Genotype ; Heterozygote ; Humans ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA ; Sulfites/metabolism* ; snRNP Core Proteins/genetics*

PURPOSE: Gastric carcinoma tissues release high level of prostaglandin E2 (PGE2) when compared to non-neoplastic mucosa, and cyclooxygenase-2 (COX-2), which is the rate-limiting enzyme in prostaglandin (PG) biosynthesis, is often overexpressed in gastric carcinomas and during gastric carcinogenesis. However, little is known about the expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the key enzyme responsible for the biological inactivation of PG, in gastric carcinomas. MATERIALS AND METHODS: We investigated the expression of 15-PGDH in 28 cases of advanced gastric carcinomas by Western blot analysis and also the relation between its expression and the gene promoter methylation. RESULTS: 15-PGDH expression was significantly decreased in gastric carcinomas compared to corresponding non-neoplastic tissues and inversely correlated with the expression of proliferating cell nuclear antigen in gastric carcinomas. However, there was no correlation between 15-PGDH expression and pathological findings such as nodal metastasis and vascular invasion. Promoter hypermethylation of 15-PGDH gene was not detected in carcinomas, with only a negligible expression of the enzyme. CONCLUSION: Our results suggested that 15-PGDH has tumor suppressor activity in gastric carcinomas.
Aged ; Base Sequence ; DNA Methylation ; DNA Primers/genetics ; DNA, Neoplasm/genetics ; Female ; Humans ; Hydroxyprostaglandin Dehydrogenases/genetics/*metabolism ; Male ; Middle Aged ; Promoter Regions, Genetic ; Stomach Neoplasms/*enzymology/genetics

OBJECTIVE: To establish a miniSTR multiplex set including three STR loci unlinked from the CODIS loci: D1S1676, D6S1274 and D17S1299, to generate amplified fragment less than 115 bp in size and to study the genotype of degraded DNA samples. METHODS: After amplification with different fluorescence labeled primers, the amplified products from 100 unrelated individual and 2 highly degraded specimens were analyzed by 310 Genetic Analyzer. RESULTS: Three miniSTR loci were determined by fluorescence-labeled multiplex-PCR technique. Each locus was successfully genotyped in all 100 samples. In D1S1676, D6S1274 and D17S1299 loci, 9, 9, 7 alleles and 27, 23, 18 genotypes were observed respectively. The distribution of genotype for three miniSTR loci in Chengdu Han population was in accordance with Hardy-Weinberg equilibrium. The combined exclusion probability and the combined discrimination power of the three STR loci in Chengdu Han population were 0.9991 and 0.9160 respectively. CONCLUSION This miniSTR multiplex set could be used in individual identification and paternity test. It also provides a new method in the analysis of degraded DNA sample.
Alleles ; China/ethnology* ; DNA/metabolism* ; DNA Fingerprinting/methods* ; DNA Primers ; Forensic Genetics/methods* ; Gene Frequency ; Genotype ; Humans ; Nucleic Acid Amplification Techniques/methods* ; Polymorphism, Genetic ; Tandem Repeat Sequences

OBJECTIVETo determine whether 3'phosphorothioate-modified-2 terminal mismatched primers can turn off DNA polymerization mediated by Exo(+) polymerase.

METHODSTwo-directional primer extension was performed using polymerase with and without 3' exonuclease activity. The effects of unmodified primers and 3' phosphorothioate-modified primers on primer extension were evaluated.

RESULTSExo(-) polymerase yielded products from matched and mismatched primers regardless of their modification. However, 3' phosphorothioate-modified primers with a single base mismatch at -2 position worked similarly to the terminal (-1) mismatched primers in triggering the novelly reported "off-switch" of Exo(+) polymerase.

CONCLUSIONThese data suggested that the "off-switch" can be of enormous application in the diagnosis of single gene diseases and in the association studies by single nucleotide polymorphism screening.

DNA Primers ; chemistry ; genetics ; Exonucleases ; metabolism ; Humans ; Phosphorothioate Oligonucleotides ; chemistry ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide

OBJECTIVETo assess the expression level of D-Tyr-tRNA(Tyr) deacylase (DTD) in SAMP8 mice and speculate the function of DTD in disorders associated with Alzheimer's disease (AD).

METHODSAltogether 12 SAMP8 mice and 12 SAMR1 mice were used in this study. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were performed to detect the mRNA and protein levels of DTD in the mice. Purified DTD protein was injected into lateral ventricle to investigate the function of DTD in SAMP mice. The behavior of the mice was tested by using a Step-through Test System.

RESULTSBoth mRNA and protein levels of DTD were found to be significantly lower in SAMP8 mice compared with those in SAMR1 mice (P<0.05). In vivo injection of DTD protein did not lead to an obvious change in behavior of SAM mice.

CONCLUSIONSDTD might function in the process of AD-associated pathology and could possibly participate in physiology process in a long-term manner to orchestrate with other regulators in order to maintain the balance of organism.

Alzheimer Disease ; enzymology ; Aminoacyltransferases ; metabolism ; Animals ; Base Sequence ; DNA Primers ; Disease Models, Animal ; Mice ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo characterize the heterotrophic nitrifying bacteria.

METHODSThe bacteria were isolated from membrane bioreactor for treating synthetic wastewater using the method newly introduced in this study. Fluorescence in situ hybridization (FISH) was used to validate the nonexistence of autotrophic ammonia oxidizers and nitrite oxidizers. Batch tests were carried out to investigate the capability of heterotrophic nitrification by the pure culture. Phylogenetic analysis of the pure culture was performed.

RESULTSA heterotrophic nitrifier, named Bacillus sp. LY, was newly isolated from the membrane bioreactor system in which the efficiency of TN removal was up to 80%. After 24-day, incubation, the removal efficiency of COD by Bacillus sp. LY was 71.7%. The ammonium nitrogen removal rate after assimilation nearly ceased by Bacillus sp. LY was 74.7%. The phylogenetic tree of Bacillus sp. LY and the neighbouring nitrifiers were given.

CONCLUSIONSThe batch test results indicate that Bacillus sp. LY can utilize the organic carbon as the source of assimilation when it grows on glucose and ammonium chloride medium accompanying the formation of oxidized-nitrogen. It also can denitrify nitrate while nitrifying. Bacillus sp. LY may become a new bacterial resource for heterotrophic nitrification and play a bioremediation role in nutrient removal.

Bacillus ; classification ; genetics ; isolation & purification ; metabolism ; Base Sequence ; DNA Primers ; DNA, Ribosomal ; genetics ; Environmental Restoration and Remediation ; methods ; Nitrates ; metabolism ; Phylogeny ; RNA, Ribosomal, 16S ; genetics

A geldanamycin (GDM) producing strain, Streptomyces hygroscopicus 17997, was isolated from Yunnan China soil by our institute researchers. GDM is an ansamycin antibiotic, which has the ability to bind with Hsp90 (Heat Shock Protein 90) and alter its function. Hsp90 is a chaperone protein involved in the regulation of the cell cycle, cell growth, cell survival, apoptosis, and oncogenesis. So it plays a key role in regulating the physiology of cells exposed to environmental stress and in maintaining the malignant phenotype of tumor cells. As an inhibitor of Hsp90, GDM possesses potent antitumor and antivirus bioactivity, but the hypato-toxicity and poor solubility in water limits its clinical use. Two GDM derivatives, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) and 17-dimethylamino-ethylamino-17-demethoxygeldanamycin (17-DMAG), both showing lesser hepato-toxicity, are now in Phase II and Phase I clinic trials. In order to accomplish the structure modification of GDM by genetic means, an attempt to obtain the biosynthetic gene cluster of GDM from S. hygroscopicus 17997 was made. In this study, a pair of primers was designed according to a conserved sequence of one of possible post-PKS (polyketides synthase) modification genes, the carbamoyltransferase (CT) gene (gdmN) in GDM biosynthesis. The 732 bp PCR product was obtained from the S. hygroscopicus 17997 genomic DNA. Through the colony-PCR Binary Search Method, using the CT gene primers, six positive cosmid clones, CT1-6, were identified from the S. hygroscopicus 17997 cosmid genomic library. The CT gene containing fragments were verified and localized by Southern blot. The CT-4 positive cosmid was then sub-cloned and sequenced. Approximately 28.356kb of foreign gene sequence from CT-4 cosmid and by further PCR extension reaction was obtained. Based on BLAST analysis, this sequence contains 13 possible ORFs and their deduced functions are believed to be involved in GDM production. The ORF1 encoding products show homology (87%) with incomplete sixth module and complete seventh module of PKS, gdmA3, in S. hygroscopicus NRRL 3602. The ORF2-13 gene products are similar to gdmF(9 5%), gdmM(8 8%), gdmN (92%), gdmH (92%), I (93%), J (90%), K (93%), G (96%), gdmO (91%), gdmP (93%) and two transcription regulation genes gdmRI (83%) and gdmRII (90%). The obtained possible GDM biosynthetic gene cluster in S. hygroscopicus 17997 will facilitate the further functional analysis of the genes and to modify the structure of GDM through combinatorial biosynthesis.
Anti-Bacterial Agents ; metabolism ; Benzoquinones ; metabolism ; Carboxyl and Carbamoyl Transferases ; genetics ; Chromosome Mapping ; Cloning, Molecular ; DNA Primers ; genetics ; Lactams, Macrocyclic ; metabolism ; Multigene Family ; Streptomyces ; genetics ; metabolism

In recent years, there have been increasing reports of KPC-producing Klebsiella pneumoniae in Korea. The modified Hodge test can be used as a phenotypic screening test for class A carbapenamase (CAC)-producing clinical isolates; however, it does not distinguish between carbapenemase types. The confirmation of type of CAC is important to ensure optimal therapy and to prevent transmission. This study applied a novel multiplex PCR assay to detect and differentiate CAC genes in a single reaction. Four primer pairs were designed to amplify fragments encoding 4 CAC families (SME, IMI/NMC-A, KPC, and GES). The multiplex PCR detected all genes tested for 4 CAC families that could be differentiated by fragment size according to gene type. This multiplex PCR offers a simple and useful approach for detecting and distinguishing CAC genes in carbapenem-resistant strains that are metallo-beta-lactamase nonproducers.
Bacterial Proteins/*genetics/metabolism ; DNA Primers/metabolism ; Databases, Genetic ; Humans ; Klebsiella Infections/microbiology ; Klebsiella pneumoniae/genetics/isolation & purification/metabolism ; *Multiplex Polymerase Chain Reaction ; beta-Lactamases/*genetics/metabolism

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

OBJECTIVETo study the expression and intracellular localization of FRZB gene in gastric cancer tissue, and to explore its significance in gastric cancer.

METHODSThe expression of FRZB in tumor tissues from 90 patients with gastric cancer and in normal gastric mucous as control were analyzed by immunohistochemistry in tissue array. FRZB expression in gastric cancer cell lines and immortalized gastric epithelial cell line GES-1 were detected by quantitative real-time PCR(Q-PCR) and Western blot. The intracellular localization of FRZB was observed by immunofluorescence staining.

RESULTSThe positive expression rate of FRZB in gastric cancer was 92.2%. FRZB expressed in gastric cancer with well differentiation was higher than that with poor differentiation.The positive rate in normal gastric mucous was 10.0% (one out of ten). By confocal microscope, FRZB localized both in cytoplasma and nucleus, especially on the nuclear membrane. The Q-PCR and Western blot results also showed that the expression of FRZB in gastric cancer cell lines was higher than that in GES-1.

CONCLUSIONSThe expression of FRZB in gastric cancer is correlated with tumor cell differentiation and tumor Lauren classification. The nuclear localization of FRZB may contribute to its function in gastric cancer formation and progression.

Biomarkers, Tumor ; genetics ; metabolism ; Cell Line, Tumor ; DNA Primers ; Female ; Gene Expression ; Glycoproteins ; genetics ; metabolism ; Humans ; Male ; Neoplasm Staging ; Stomach Neoplasms ; genetics ; metabolism ; pathology