Development of a 2D-HPLC for simultaneous determination of rhynchophylline， isorhynchophylline， corynoxeine and isocorynoxeine / 四川精神卫生

ObjectiveTo develop a two-dimensional high-performance liquid chromatography （2D-HPLC） for simultaneous determination of the contents of four kinds of Uncaria alkaloids： rhynchophylline， isorhynchophylline， corynoxeine and isocorynoxeine. MethodsThe 2D-HPLC apparatus was comprised of a first chromatographic column in version Aston SC2 （3.5 mm×25 mm， 5 μm）， an intermediate column in version Aston SH C18 （3.5 mm×10 mm， 5 μm）， and an analytical column in version Aston SCB （4.6 mm×125 mm， 5 μm）. The mobile phase of the first and second liquid chromatography system were CAA-1 and mixed mobile phase （V BPI-1 basic mobile phase ∶ V MPI-1 mobile phase ∶ V OPI-1 organic mobile phase = 45∶14∶41）. The chromatographic parameters included a flow rate of 1.0 mL/min， a column temperature of 40℃， a wavelength of 254 nm， an injection volume of 500 μL and a detection time of 9.5 min. ResultsThe linear ranges of rhynchophylline， isorhynchophylline， corynoxeine and isocorynoxeine were 9.77~10 000.00 ng/mL （r=0.999 6）， 10.74~11 000.00 ng/mL （r=0.999 7）， 10.74~11 000.00 ng/mL （r=0.999 7）， 10.74~11 000.00 ng/mL（r=0.999 6）， respectively. The relative standard deviation （RSD） of precision， stability and repeatability were all less than 5.00%. The accuracy was 95.20%~104.01%， and the recovery rate was 93.63%~101.38%. ConclusionThe 2D-HPLC developed for simultaneous determination of four kinds of alkaloids in Uncaria is simple and accurate， which can be used as a new method for quality control of Uncaria.

Development and preliminary application of PCR-DHPLC assays for detection of the Torque Teno virus / 中国人兽共患病学报

In order to identify the Torque Teno virus (TT virus),a PCR-DHPLC assay was performed in this study.Primers specific were selected according to the characteristics of TT virus nucleic acid sequence to conduct PCR,and PCR products assayed by DHPLC.We analyzed the sensitivity,specificity,repeatability of PCR-DHPLC and applied it preliminarily on clinical detection.The specific testing was performed with TTV,HBV,HCV and HEV,no cross reaction were found,and the PCR-DHPLC assays we developed had good specification and nice repeatability.Sensitivity analysis showed that the developed PCR-DHPLC assays could detect 1.0× 101 copy/μL.Then we detected 32 serum samples by this method,real-time PCR and normal PCR at same time.The results showed that 17 TTV positives results could be observed by PCR-DHPLC for 32 samples,it is consistent with real-time PCR test results and 15 positive by normal RT-PCR.PCR-DHPLC assays showed nice specification,sensitivity,repeatability,and could be used in epidemiological investigation.

Chemotherapy drugs affect terminal lung cancer EGFR mutation status / 中国基层医药

Objective To compare the advanced non -small cell lung cancer (NSCLC)patients before and after chemotherapy peripheral blood EGFR mutation status,we understand whether the chemotherapy drug impacts the EGFR mutation status of the advanced NSCLC patients,so as to improve the precision of EGFR TKIs -drug use. Methods To collect the peripheral blood of 30 cases of advanced NSCLC before chemotherapy and after chemotherapy for 6 cycles.DHPLC technique was used to detect the EGFR mutation states of EGFR exon 19 and in exon 21.Results In 30 patients,chemotherapy prior EGFR mutation positive rate was 53.3% (16 /30).After 6 cycles of chemotherapy, the EGFR mutation positive rate was 36.6% (11 /30),the consistent rate was 56.6 (17 /30)before and after chemo-therapy,inconsistent rate was 53.4% (13 /30).10 cases from positive to negative before chemotherapy,3 cases from negative into positive before chemotherapy with statistical significance (P =0.046).Six EGFR19 exons changed, change rate of 20%,8 EGFR21 exons shift changed at a rate of 26%.EGFR19,21 shift in 1 case,with no statistical significance(P =0.39,P >0.05).Conclusion (1)The late NSCLC patients before and after peripheral blood of EGFR mutation status change,so before starting the targeted therapy we must recive real -time detection of peripheral blood EGFR mutation status,so as to decide whether to choose EGFR TKIs targeted drug therapy.(2)EGFR21 exons transformation rate is higher than EGFR19 exons conversion rate,but with no statistical difference,this phenomenon may be related to EGFR19 exons patients who with EGFR mutations -TKIs treatment efficiency is higher.

Determination of diclofenac sodium in human plasma based on 2D-HPLC coupled with trapping column and its application / 中国药学杂志

OBJECTIVE: To establish a method for determination of diclofenac sodium in human plasma under high-fat meal condition using two-dimensional liquid chromatography (2D-HPLC) coupled with trapping column, and to evaluate the bioequi valence and the bioavailability of diclofenac sodium sustain-released tablets in healthy volunteers. METHODS: Under fed state, eighteen healthy male volunteers were divided into two groups by an open, randomized two period crossover design with a single dose of diclofenac sodium sustain-released tablets. The plasma concentrations were determined by 2D-HPLC method, and calculated pharmacokinetic parameters and bioavailability. RESULTS: The main pharmacokinetic parameters of test and reference preparations after a single dose were: AUC0-16 was (2 591.6 ± 705.8) and (2 588.8 ± 772.0) ng · h · mL-1; AUC1→∞ was (2 896.4 ±839.7) and (2 700.3 ± 806.1) ng · h · mL-1; tmax was (4.6 ± 0.7) and (4.4 ± 0.9)h; ρmax was(1 332.8 ± 912.5) and (1 325.7 ± 706.3) ng · mL-1, respectively. The relative bioavailability of test preparation in single dose was (102.4 ± 15.1)%. CONCLUSION: The 2D-HPLC method coupled with trapping column is a simple, rapid and specific for determination of diclofenac sodium in human plasma. The results of statistical analysis indicate that the two preparations are bioequivalent in healthy volunteers with a single dose under high-fat fed condition.

Métodos de investigación molecular en la detección de mutaciones germinativas en cáncer colorrectal hereditário / Molecular research methods in the detection of germinal mutations in hereditary colorectal cancer

El cáncer colorrectal (CCR) es una de las principales causas de muerte por neoplasia en países sudamericanos. Las formas hereditarias de CCR son, poliposis adenomatosa familiar (FAP) y cáncer colorrectal hereditario sin poliposis (HNPCC) o Síndrome de Lynch (SL), que es la forma más común. La detección de mutaciones en los genes de reparo de ADN (MMR) y en el gen APC permite el desarrollo de estrategias de prevención. Algunas de estas metodologías de diagnóstico molecular son aplicadas para la investigación y detección de mutaciones en estos genes, tales como el Test de la Proteína Truncada (PTT), Análisis de polimorfismos de conformación de cadena simple (SSCP), Cromatografia liquida desnaturante de alta performance (DHPLC) y Reacción en Cadena de la Polimerasa (PCR) en tiempo real (qPCR).

Colorectal cancer (CRC) is one of the main causes of death in South American countries. The hereditary forms of CRC are, familial adenomatous (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch Syndrome (LS), which is the most common form. The detection of mutations in the DNA repair genes (MMR) and in the APC genes enables the development of prevention strategies. Some of these methods for molecular diagnosis are applied in research and the detection of mutations of these genes, such as the partial thromboplastin time test (PTT), the single strand conformational polymorphism test (SSCP), the Denaturing High Performance Liquid Chromatography test (DHPLC) and the Polymerase Chain Reaction (PCR) in real time (qPCR).

Estudo do gene GPB2 no carcinoma de células escamosas do esôfago esua relação com a proteína P53

O câncer de esôfago está entre os 10 tipos de câncer mais incidentes no mundo e no Brasil. O carcinoma de células escamosas do esôfago (CCEE) é o tipo histopatológico mais frequente e, em geral, é diagnosticado em estágios avançados, impossibilitando o tratamento curativo. A sobrevida é de menos de 10% dos pacientes após 5 anos do diagnóstico da doença. Para reverter esta situação é fundamental o entendimento de como ocorre a carcinogênese molecular esofagiana. Mutações no gene supressor de tumor TP53 são frequentes no CCEE, sendo relatados na literatura 30-70% de casos mutados. Outra alteração, recentemente descrita por nosso grupo, é o aumento da expressão da proteína ligadora de guanilato-2, GBP2, no tecido tumoral em relação ao epitélio normal adjacente de pacientes com CCEE. Dados de nosso grupo também demonstram a regulação de GBP2 dependente de p53 em uma linhagem celular de CCEE. O presente estudo, portanto, teve como objetivo geral investigar a relação entre GBP2 e p53 no CCEE através de duas etapas: pré-clínica e clínica. O objetivo pré-clínico foi investigar a possível ligação direta da proteína p53 selvagem à região promotora de GBP2 na linhagem TE-1 (CCEE, p53Met272Val) através da bioinformática e do ensaio de imunoprecipitação de cromatina. Foi identificada a ligação direta de p53 selvagem à região promotora de GBP2. Na etapa clínica, foram analisadas amostras de RNA e DNA dos tecidos tumoral e não-tumoral adjacente de pacientes com CCEE. Utilizando a técnica de PCR quantitativa, foi visto em 69,3% (18/26) dos pacientes um aumento da expressão de GBP2 no tecido tumoral em relação ao tecido não- -tumoral adjacente. O rastreamento por alterações nos éxons 5 ao 9 do gene TP53 foi realizado pela técnica de cromatografia líquida desnaturante de alta performance (dHPLC) e pela técnica de polimorfismo conformacional de fita simples (SSCP) seguido pelo sequenciamento automático. Em 56,1% (23/41) dos casos, a mutação em TP53 foi encontrada e em 3 pacientes foram encontrados polimorfismos neste gene. Não foi encontrada qualquer associação com significância estatística entre a expressão de GBP2, o status mutacional de TP53 e os dados clínico-patológicos no grupo de pacientes estudados. Os resultados deste trabalho sugerem que GBP2 é um gene-alvo de p53, resultado esse inédito na literatura, e que GBP2 tem um papel na carcinogênese do esôfago. Além disso, foi observada a ausência de associação entre a expressão de GBP2 e o status mutacional da proteína p53 durante o...

Esophageal cancer is one of the 10 most common incident cancers in the world and in Brazil. Esophageal squamous cell carcinoma (ESCC) is the most frequent histopathological type. In general, ESCC is diagnosed in advanced stages, when curative treatment is not possible. After 5 years of diagnosis, overall survival is less than 10% of patients. To change this situation, it is essential to understand how esophageal molecular carcinogenesis occurs. Mutations in TP53 tumor suppressor gene are frequent in ESCC, appearing in 30-70% of cases according to the literature. Another alteration, recently described by our group, is the increase of guanylate binding protein-2, GBP2, expression in tumoral tissue compared to adjacent normal epithelium of patients with ESCC. Data from our group also show p53-dependent GBP2 regulation on a ESCC cell line. The present study, therefore, had as general objective investigate the relationship between GBP2 and p53 through two parts: preclinical and clinical. The pre-clinical objective was to investigate a possible direct binding of wild type p53 protein to GBP2 promoter region on TE-1 cell line (ESCC, p53Met272Val) through bioinformatics and chromatin immunoprecipitation assay. In this study, it was identified the direct binding of wild type p53 to GBP2 promoter region. On the clinical part of the study, RNA and DNA samples of tumoral and adjacent nontumoral tissues from patients with ESCC were analysed. Through quantitative PCR technique, an increase of GBP2 expression in tumoral tissue in relation to adjacent non-tumoral tissue was seen in 69.3% (18/26) of patients. The screening of alterations on exons 5 to 9 of TP53 gene was performed by denaturing high performance liquid chromatography (dHPLC) and followed by automated sequencing. In 56,09% (23/41) of cases, TP53 mutation was found and in 3 patients polymorphisms were found. No statistically significant association was found between GBP2 expression, TP53 mutational status and clinicopathological data on the studied group. These results suggest that GBP2 is a p53 target-gene, an unpublished data on the literature, and that GBP2 has a role on esophageal carcinogenesis. Also, it was found no association between increased GBP2 gene expression and the presence of wild type p53 during esophageal tumor development. These data encourage a better characterization of GBP2 on ESCC to better understand its participation on the development of this cancer.

Detection of Rifampin Resistant Mycobacterium tuberculosis complex using Denaturing HPLC / 대한진단검사의학회지

BACKGROUND: Tuberculosis (TB) remains an important cause of morbidity and mortality throughout the world. The surge of TB has been accompanied by an increase in multi-drug-resistant tuberculosis (MDR-TB). In this study, we developed a denaturing HPLC (DHPLC) method for detecting rpoB gene mutation as a rifampin resistance based on sequence. METHODS: In this study, we used 99 mycobacterial isolates grown in Ogawa media. At first, we used a PCR method that can amplify the 235 bp and 136 bp rpoB DNAs of Mycobacterium tuberculosis complex (MTB) and Non-tuberculous mycobacteria (NTM). And then, PCR-restriction fragment length polymorphism (RFLP) of rpoB DNA (342 bp), which comprises the Rif(T) region, was used for the differential identification of Mycobacteria. Finally, we detected these amplicons by DHPLC, compared to PCR-RFLP results, and performed sequencing. RESULTS: Among 99 mycobacterial isolates, 80 (81%) were MTB and 19 (19%) were NTM. NTM were identified to 7 different species by DHPLC and PCR-RFLP. rpoB mutation was detected in 9 (11%) of the MTB specimens. These results were confirmed by using sequencing. CONCLUSIONS: DHPLC provided a rapid, simple, and automatable performance for detection of rifampin resistant Mycobacterium tuberculosis complex and would be helpful as a supplemental method in high-throughput clinical laboratories.

Variaciones genéticas del gen supresor de tumores TP53: relevancia y estrategias de análisis / Genetic variations of the tumor suppressor TP53: Outstanding and strategies of analysis

In the modern society, cancer remains an important cause of death. Cancer development is a very complex process that involves alterations in genes regulating cellular growth. Among these alterations or variations, are included point mutations, genetic susceptibility by single nucleotide polymorphisms or "SNP" and alteration or loss in tumor suppressor genes functions. The tumor suppressor TP53 is one of the most important and studied genes on cancer genetics. Therefore, it has been demonstrated that TP53 present mutations in more than 50% of all types of human cancer and encodes a multifunctional protein whose absence contributes to genomic instability, the accumulation of mutations and increased tumor development. The identification of such alterations in cancerous cells at level of single nucleotide is very important, because its implication in the loss or alteration in the function of this gene, its clinical relevance and finally, its association with response to therapy and prognosis. Due to the large interesting issue, in this work we are focused only in two of the most common genetic variations present in this gene: the point mutations and SNP remarking some outstanding molecular characteristics needed for design its analysis.

El cáncer continúa siendo una importante causa de muerte en la sociedad moderna. Los procesos en el desarrollo del cáncer son muy complejos e involucran alteraciones en genes implicados en la proliferación celular. Entre estas alteraciones o variaciones genéticas se incluyen las mutaciones puntuales, la susceptibilidad genética por polimorfismos de un solo nucleótido o "SNP", así como la pérdida o alteración en la función de genes supresores de tumores. El gen supresor de tumores TP53 es uno de los genes más importantes y estudiados en la genética del cáncer, ya que se encuentra mutado en más del 50% de todos los tipos de cáncer humano y codifica para una proteína multifuncional cuya deficiencia contribuye a la inestabilidad genómica que conduce a la acumulación de mutaciones y a la aceleración en el desarrollo del tumor. Es importante el estudio de dichas alteraciones genéticas presentes en las células cancerosas que puedan ser detectadas a nivel de un solo nucleótido, por su implicación en la pérdida o alteración en la función del gen TP53, así como por la relevancia clínica que ellas puedan tener al ser asociadas a la respuesta de una terapia particular o al pronóstico. Debido a la extensión de este trabajo solamente revisaremos dos de las variaciones genéticas importantes en este gen: las mutaciones puntuales y los SNP, haciendo ánfasis en algunas características moleculares que son relevantes en el diseño de estrategias de análisis para su detección.

Screening of SNPs in the coding region of PRKCG via DHPLC and study of their association with Parkinson's disease / 中国康复理论与实践

@#ObjectiveTo screen the variations of the human protein kinase Cγ gene (PRKCG) and study their association with Parkinson's disease(PD).MethodsDNA was extracted from blood of patients with PD and matched normal controls. All 18 exons including the exon-intron junctions were amplified in 17 different PCR fragments, which were analyzed for the presence of variations by DHPLC. The PCR products with a heteroduplex peak were sequenced. Significance was evaluated from 2×2 contingency tables byX2 test on the basis of the total number of alleles at each locus. Case-control association analysis was performed between candidate polymorphisms and PD. ResultsIn the 50 early-onset PD(EOPD) patients and 50 controls, there was no missense mutation, insertions or deletions in coding regions of the PRKCG. But 2 different single nucleotide polymorphism(SNPs) in exons, 5 different SNPs and 1 tetranucleotide repeat in introns were identified. Five of them [IVS3+96G>T, IVS11+26T>G, IVS15-41T>C, IVS16-59G>A, IVS16-42(TCTG)1-2] were described here for the first time. Three of them (IVS11+26T>G, IVS13+76T>C,1497T>C),in complete linkage,constituted a haplotype block. In the preliminary association analysis, the frequency of IVS13+76C, IVS11+26G and 1497C allele on this haplotype block was significantly higher in EOPD patients than the controls (24% vs 9%)(X2=8.165,P=0.004,OR=3.193, 95%CI:1.400-7.282). But in a larger sample of 156 EOPD patients, 153 late-onset PD(LOPD) patients and 195 normal controls, there was no significant difference between the three groups (12.8%,13.7% ,14.6%)(X2=0.471,P=0.790). ConclusionThe PRKCG gene might not be a risk factor for sporadic PD.

Mutational Analysis of GABAA Receptor gamma2 Subunit Gene, GABRG2, in Patients with Febrile Seizure or Generalized Epilepsy with Febrile Seizure Plus / 대한소아신경학회지

PURPOSE: The GABAergic system has long been implicated in epilepsy with defects in GABA neurotransmission linked to epilepsy in both experimental animal models and human syndromes. Recently, mutations in the GABA(A) receptor gamma 2 subunit (GABRG2) gene were identified in two families with generalized epilepsy with febrile seizures plus(GEFS+) and two families with childhood absence epilepsy and febrile seizures. We tested the hypothesis that genetic variations in the GABRG2 gene confer susceptibility to febrile seizure in the Korean population. METHODS: A total of 22 febrile seizure patients with or without afebrile seizures were selected. To identify unknown mutations in GABRG2 gene, a total of nine exons were amplified and screened by DHPLC method. DNA fragments showing abnormal DHPLC elution patterns were subsequently sequenced. RESULTS: Among 22 febrile seizure patients, 5 patents(23%) were familial and 7 patients were sporadic cases. And 17(77%) experienced afebrile seizures and 5 patients didn't. Seizure types of 17 febrile patients with afebrile seizure were 13 idiopathic generalized epilepsies, 1 juvenile myoclonic epilepsy, 1 childhood absence epilelsy and 2 complex partial seizures. We identified two single nucleotide polymorphisms in exon 1 and exon 3. In exon 1, C69C/T polymorphism(dsSNP:3219203) was identified in 4 patients, which had already been reported. In exon 3, C541C/T polymorphism was identified in nine patients and eight patients showed C/T hetero forms and one patient showed T/T homo mutant form. This C541C/T allelic variations were novel and identified in febrile seizure patients with afebrile seizures. But this variation didn't show significant correlations with febrile seizure patterns or family history of patients. CONCLUSION: Our study identified two exonal polymorphisms and one is novel. The GABRG2 gene seems to confer a rare rather than a frequent major susceptility effect to febrile seizure.

Mutational Analysis of Growth Differentiation Factor-9 Gene in Korean Women with Premature Ovarian Failure / 대한산부인과학회잡지

The clinical models for studying ovary-determining genes may be premature ovarian failure (POF). POF is a condition causing amenorrhea, hypoestrogenism, and elevated gonadotropins in women under 40 years old. FSH receptor, LH receptor, inhibin, GDF-9 (growth differentiation factor-9), BMP-15 (bone morphogenetic protein-15), DIAPH2 (diaphanous gene) and XPNPEP2 (X-prolyl aminopeptidase) genes were proposed as a possible candidate gene, but until recently, only mutations in FSH receptor, LH receptor and inhibin genes have been identified in POF patients. Therefore mutation screening of another POF gene necessary to reveal the principal causative genes of POF. OBJECTIVE: The present study was performed to analyze the mutation of GDF-9 gene in Korean patient with POF and to investigate whether mutation of these gene is a likely main cause of POF. METHODS: Eighty-six women with POF were studied and thirty-six normal women were enrolled as control. Mutation screening of these genes were performed by denaturing HPLC and were confirmed by automatic sequencing. RESULTS: Three different mutations of GDF-9 gene were identified in Korean women with POF; Arg3Cys mutation in one patient, Leu40Val mutation in one patient, Asp57Tyr mutation in 10 patients and 5 normal controls. Arg3Cys mutation and Leu40Val mutation were likely cause of disease. Frequencies of Arg3Cys mutation and Leu40Val mutation were 1.2%, respectively. Asp57Tyr mutation was common polymorphism in Korean women. All mutations was a novel mutation found in the present study. CONCLUSION: POF was resulted by mutations of GDF-9 gene, but mutations of GDF-9 gene are not likely main causes of POF because of low frequency of mutations.

Application of DHPLC in molecular diagnosis of X-linked adrenoleukodystrophy(report of 12 cases) / 解放军医学杂志

T),one dinucleotide deletion(1801-02 del AG) and one base insertion(1125 ins GCCATCG),which resulted in eight missense mutations,two nonsense mutations and two frame shift mutations,namely P534R,G343V,R259W,A141T,R401Q,K276E,Y174C,A314P,S108X,Q177X,fs E471 and fs A247.Conclusion The combined DHPLC and sequencing approach may act as a rapid and efficient method for ABCD1 gene mutation analysis in patients and carriers of X-linked adrenoleukodystrophy families.There exist different ABCD1 gene mutations in different pedigrees,and no obvious correlation between the genotype and phenotype has been found.