Fingerprint study of Perns et Testis Cervi mtDNA with PCR-SSCP / 中国药学杂志

OBJECTIVE: To establish a method of molecular biology to identify the authenticity and varieties of Penis et Testis Cervi rapidly, simply and accurately. METHODS: The mitochondrial DNAs (mtDNA) of dried Cervus nippon Penis, Cervus elaphus. L Penis, Penis et Testis Bovis and commercial products were extracted and purified with column chromatography. A pair of primers were designed for PCR special amplification of mtDNA according to cytochrome b gene sequence in the cervidae mtDNA GenBank; the products of PCR were analyzed for the single strand conformation polymorphism (SSCP) by denaturing polyacrylamide gel electrophoresis (PAGE). RESULTS: The positive control samples and some commercial products showed clear single strand DNA (ssDNA) bands with different mobility, but the negative control sample and some other commercial products showed nothing. CONCLUSION: Column chromatography method is a good way for getting mtDNA with high purity and less destruction of structure for the follow-up special PCR amplification; SSCP technique not only shows ssDNA bands of mutually complementing clearly, but also reveals the difference of mobility among ssDNAs from all samples directly. For that reason, the technique of PCR-SSCP will be reliable and feasible to indentify the authenticity and varieties of products from cervidae.

O Polimorfismo no códon 98 do gene de galectina-3 não está associado a tumores benignos e malignos de tiróide / Polymorphism on codon 98 of the galectin-3 gene are not associated to benign and nalignant thyroid tumors / Polymorphism on codon 98 of the galectin-3 gene are not associated to benign and nalignant thyroid tumors

Galectina-3 é uma proteína multifuncional altamente expressa em câncer de tiróide. O gene de galectina-3 (LGALS3) apresenta vários candidatos a SNPs anotados, no entanto a relação entre estes SNPs e variações fenotípicas específicas relevantes à saúde não foi avaliada. Neste estudo, investigamos SNPs do LGALS3 e uma possível associação destes com a tumorigênese tiroidiana. A presença de SNPs do LGALS3 em linhagens de carcinoma de tiróide (WRO, NPA, TPC-1, ARO), tecidos tiroidianos de 55 pacientes com diagnóstico de bócio multinodular ou carcinoma papilífero e linfócitos do sangue periférico de 45 indivíduos saudáveis foi avaliada por seqüenciamento e SSCP. A análise da seqüência codificadora do LGALS3 mostrou que o sítio T98P apresenta uma grande variação genotípica, visto que observamos os padrões homozigoto (AA ou CC) e heterozigoto (AC). Em linhagens de carcinoma de tiróide, o genótipo da NPA no sítio T98P do LGALS3 é CC, enquanto TPC-1, WRO e ARO são AC. As freqüências genotípicas do T98P do LGALS3 observadas em bócio multinodular (AC= 67 por cento, AA= 23 por cento, CC= 10 por cento) e carcinoma papilífero (AC= 68 por cento, AA= 20 por cento, CC= 12 por cento) foram semelhante à freqüência observada na população controle (AC= 60 por cento, AA= 24 por cento, CC= 16 por cento). Em conclusão, não observamos associação entre o genótipo T98P do LGALS3 e o fenótipo de tumor benigno ou maligno de tiróide.

Galectin-3 is a multifunctional protein highly expressed in thyroid cancer. The galectin-3 gene (LGALS3) has several annotated candidates SNPs, however the relationship between galectin-3 SNPs and specific phenotypic variations relevant to health has not been evaluated. In this study, we investigated SNPs in the galectin-3 gene and a putative association with thyroid tumorigenesis. The presence of LGALS3 SNPs in thyroid carcinoma cell lines (NPA, TPC-1, WRO, ARO), thyroid tissues of 55 patients with multinodular goiter or papillary carcinoma diagnosis and lymphocytes of peripherical blood of 45 healthy individuals was evaluated by sequencing and SSCP. The analysis of LGALS3 coding sequence showed that the T98P site presents a great genotypic variation, since we observed both homozygous (AA or CC) and heterozygous (AC) patterns. In thyroid carcinoma cell lines, the genotype of NPA in the LGALS3 T98P site is CC, while TPC-1, WRO and ARO are AC. The genotypic frequency of T98P SNP observed in multinodular goiter (AC= 67 percent; AA= 23 percent; CC= 10 percent) and papillary carcinoma (AC= 68 percent; AA= 20 percent; CC= 12 percent) were similar to the frequency observed in the control population (AC= 60 percent, AA= 24 percent, CC= 16 percent). In conclusion, no association between LGALS3 T98P genotype and the phenotype of the benign or malignant thyroid tumor was observed.

Relationship Between gyrA Gene Mutations in Clinical Isolates of Pseudomonas Aeruginosa and Quinolone Resistance / 中国药房

OBJECTIVE:To observe the relationship between gyrA gene mutations of the clinical isolates of Pseudomonas aeruginosa and quinolone resistance and to evaluate the feasibility of analyzing gyrA gene mutations using PCR-RFLP-SSCP.METHODS:With gyrA gene order of the clinical isolates of Pseudomonas aeruginosa taken as target sequence,gyrA gene mutations in strain ATCC 27853 and 16 clinical isolates of Pseudomonas aeruginosa were analyzed contrastively using PCR,PCR-RFLP,PCR-SSCP,and DNA sequencing.RESULTS:Of the total 8 ciprofloxacin resistant Pseudomonas aeruginosa,6 strains showed single point ACC→ ATC mutation in the gyrA gene at codon 83,leading to amino acid substitution of Thr83→Ile.SacⅡ digestion fragment of the PCR amplified products in gyrA gene was in line with the sequencing results.SSCP showed that the banding patterns of all strains were different from that of strain ATCC 27853 except 2 strains.CONCLUSION:The molecular mechanism of the quinolone resistant Pseudomonas aeruginosa isolated from clinics was manifested as mutations in the gyrA gene at codon 83.The results showed that PCR-RFLP-SSCP is a rapid and accurate method for the detection of basyl variation in gyrA in quinolone resistant Pseudomonas aeruginosa.

Molecular Analysis of Oculocutaneous Albinism Patients in Korea

BACKGROUND: Oculocutaneous albinism (OCA) is a genetic disorder of the melanin pigment system in which melanin synthesis is reduced or absent in the skin, hair, and eyes. OCA is classified into two major types, and tyrosinase-related OCA can be produced by mutations of the structural gene for tyrosinase enzyme (TYR gene). OBJECTIVE: The purpose of this study was to analyze the segregation of mutant alleles of the TYR gene in tyrosinase-negative and tyrosinase-positive Korean OCA patients and families. METHODS: We amplified exon I, II, and III of the TYR gene of Korean OCA patients and their families by polymerase chain reactions (PCR), and analyzed the mutations by restriction fragment length polymorphism (RFLP) analysis in exon I and single-strand conformation polymorphism (SSCP) analyses in exon II and exon III. RESULTS: Two tyrosinase-negative cases showed mutations in exon I. Four tyrosinase-nega-tive cases and one tyrosinase-positive case showed mutations in exon II, and one tyrosinase-neg- ative case showed mutations in exon III. In summary, we found three kinds of mutation in four tyrosinase-negative OCA patients and one tyrsinase-positive OCA patient. CONCLUSIONS: RFLP and SSCP analysis can provide a basis for a rapid and sensitive screening system to detect TYR gene mutations of Korean OCA patients and their families.