RESUMO
Inappropriate c-MET signaling in cancer can enhance tumor cell proliferation, survival, motility, and invasion. Inhibition of c-MET signaling induces apoptosis in a variety of cancers. It has also been recognized as a novel anticancer therapy approach. Furthermore, reports have also indicated that constitutive expression of P-glycoprotein (ABCB1) is involved in the HGF/c-MET-related pathway of multidrug resistance ABCB1-positive human hepatocellular carcinoma cell lines. We previously reported that elevated expression levels of PKCδ and AP-1 downstream genes, and HGF receptor (c-MET) and ABCB1, in the drug-resistant MES-SA/Dx5 cells. Moreover, leukemia cell lines overexpressing ABCB1 have also been shown to be more resistant to the tyrosine kinase inhibitor imatinib mesylate. These findings suggest that chemoresistant cancer cells may also develop a similar mechanism against chemotherapy agents. To circumvent clinical complications arising from drug resistance during cancer therapy, the present study was designed to investigate apoptosis induction in ABCB1-overexpressed cancer cells using c-MET-targeted RNA interference technology in vitro and in vivo. The results showed that cell viability decreased and apoptosis rate increased in c-MET shRNA-transfected HGF/c-MET pathway-positive MES-SA/Dx5 and MCF-7/ADR2 cell lines in a dose-dependent manner. In vivo reduction of tumor volume in mice harboring c-MET shRNA-knockdown MES-SA/Dx5 cells was clearly demonstrated. Our study demonstrated that downregulation of c-MET by shRNA-induced apoptosis in a multidrug resistance cell line.
Assuntos
Proteínas Proto-Oncogênicas c-met/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/biossíntese , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Apoptose/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos , Feminino , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Células MCF-7 , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Proto-Oncogênicas c-met/deficiência , Proteínas Proto-Oncogênicas c-met/metabolismo , Sarcoma/tratamento farmacológico , Sarcoma/genética , Sarcoma/metabolismo , Sarcoma/patologia , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Thymidylate synthase (TS) is an important target for chemotherapeutic treatment of cancer. However, efficacy of TS-targeted anticancer drugs is limited by the development of drug resistance as a result of TS gene amplification. In this work, a phosphorothioated antisense oligonucleotide (ODN), designated ATS-2, was used to suppress cellular synthesis of TS. ATS-2 at 0.2 microM concentration was mixed with lipofectin in a charge ratio of 1:1 and was used to treat the human embryonic kidney (HEK) cell line. A reduction of TS mRNA and protein was achieved. Furthermore, a dose-dependent reduction of cumulative viable cells of up to 98% was observed. Flow cytometer analysis of cell cycle progression indicates that ATS-2-treated cells were arrested and went into apoptosis at the S phase, possibly because of thymidine shortage, suggesting that ATS-2 is specifically effective for dividing cells. When used in combination with the anticancer drug FdUrd, ATS-2 exerted a additive inhibitory effect on cellular proliferation. To elucidate the possible role of cellular thymidine kinase (TdR kinase) in ATS-2 treatment, a second cell line, HeLa, was used. Both HEK and HeLa have similar rates of cell division and ODN uptake. In contrast to HEK, which was shown to have very low levels of TdR kinase activity in [(3)H]thymidine incorporation experiments, [(3)H]thymidine incorporation in HeLa was 15-fold greater than that of HEK. We found that HeLa cells were sensitive to FdUrd but were rather resistant to ATS-2. On the contrary, HEK cells were sensitive to ATS-2 but insensitive to FdUrd. Effects of ATS-2 and FdUrd are, therefore, complementary in thymineless treatment too.
Assuntos
Apoptose , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Fase S/efeitos dos fármacos , Timidilato Sintase/antagonistas & inibidores , Antimetabólitos Antineoplásicos/farmacologia , Transporte Biológico/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Portadores de Fármacos , Interações Medicamentosas , Floxuridina/farmacologia , Células HeLa , Humanos , Fosfatidiletanolaminas , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Timidilato Sintase/genéticaRESUMO
BACKGROUND: Spinocerebellar ataxia (SCA) is a heterogeneous group of neurodegenerative disorders. The mutational basis for most of these disorders is an expanded CAG repeat sequence within the coding regions of the genes involved. The prevalence of SCA in the ethnic Chinese on Taiwan remains unclear. Moreover, there has been no report of SCA type 6 (SCA6) among Chinese people. OBJECTIVES: To characterize the prevalence of SCA in the ethnic Chinese on Taiwan, and to specifically characterize Chinese patients with SCA6 in terms of clinical and molecular features. PATIENTS AND METHODS: Using a molecular approach, we investigated SCA in 74 Taiwanese families with dominantly inherited ataxias and in 49 Taiwanese patients with sporadic ataxias. Clinical and molecular features of SCA6 were further characterized in 12 patients from 8 families and in 2 sporadic cases. Furthermore, the intragenic polymorphic marker D19S1150 was amplified by polymerase chain reaction to analyze for linkage disequilibrium. RESULTS: Machado-Joseph disease-SCA3 was the most common type of autosomal dominant SCA in the Taiwanese cohort, accounting for 35 cases (47.3%), followed by SCA6 (8 [10.8%]), SCA2 (8 [10.8%]), SCA1 (4 [5.4%]), SCA7 (2 [2.7%]), dentatorubropallidoluysian atrophy (1 [1.4%]), and SCA8 (0%). The genes responsible for 16 (21.6%) of Taiwanese dominantly inherited SCA cases remain to be determined. Among the 49 patients with sporadic ataxias in the present series, 2 (4.1%) were found to harbor SCA6 mutations. In the families with SCA6, we found significant anticipation in the absence of genetic instability on transmission, indicating that some other mechanism might account for the anticipation. The same frequent allele of the intragenic DNA marker (D19S1150) was shared by 7 of 10 Taiwanese families with SCA6. CONCLUSIONS: Although SCA6 has, so far, not been reported in mainland Chinese, we found a geographic cluster of families with SCA6 on Taiwan. Genotyping studies suggest a founder effect in the Taiwanese patients with SCA6.
Assuntos
Ataxia Cerebelar/genética , Efeito Fundador , Genes Dominantes/genética , Ataxias Espinocerebelares/genética , Adulto , Alelos , Ataxia Cerebelar/etnologia , China/etnologia , Feminino , Heterozigoto , Humanos , Doença de Machado-Joseph/genética , Masculino , Pessoa de Meia-Idade , Ataxias Espinocerebelares/etnologia , Taiwan/epidemiologia , Repetições de TrinucleotídeosRESUMO
Progress in the understanding of early mammalian embryo development has been severely hampered by scarcity of study materials. To circumvent such a constraint, we have developed a strategy that involves a combination of in silico mining of new genes from expressed sequence tags (EST) databases and rapid determination of expression profiles of the dbEST-derived genes using a PCR-based assay and a panel of cDNA libraries derived from different developmental stages and somatic tissues. We demonstrate that in a random sample of 49 independent dbEST-derived zinc finger protein genes mined from a mouse embryonic 2-cell cDNA library, more than three-quarters of these genes are novel. Examination of characteristics of the human orthologues derived from these mouse genes reveals that many of them are associated with human malignancies. Expression studies have further led to the identification of three novel genes that are exclusively expressed in mouse embryos before or up to the 8-cell stage. Two of the genes, designated 2czf45 and 2czf48 (2czf for 2-cell zinc finger), are zinc finger protein genes coding for a RBCC protein with a RFP domain and a protein with three C2H2 fingers, respectively. The third gene, designated 2cpoz56, codes for a protein with a POZ domain that is often associated with zinc finger proteins. These three genes are candidate genes for regulatory or other functions in early embryogenesis. The strategy described in this report should generally be applicable to rapid and large-scale mining of other classes of rare genes involved in other biological and pathological processes. Mol. Reprod. Dev. 59:249-255, 2001.
Assuntos
Bases de Dados Factuais , Embrião de Mamíferos/fisiologia , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Dedos de Zinco/genética , Animais , Biblioteca Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodosRESUMO
A number of genotypes of the human papillomaviruses (HPV) are associated with malignancies of the uterine cervix. Sequencing work has revealed the existence of intratype HPV variants with minor differences in the nucleotide sequence. More recent data suggest the possibility that some of the variants may have different modes of clinical manifestation. In this study, sequences of the E6 and E7 oncogenes of 17 HPV16 isolates derived from PAP smear samples of Taiwanese patients were analyzed. A number of E6 and E7 novel variants were found. Particularly, a prevalent (64.7%) E6 polymorphic site A442C with an E113D amino acid substitution seems specific to Taiwanese patients. In E7, two novel but silent polymorphic sites G663A (41.2%) and T846C (88.2%) were also prevalent in the samples analyzed. The yeast two-hybrid system was adopted for rapid assessment of relative E7-pRb binding affinity in the variants. The relative binding affinities of the E7 proteins of different HPV types to pRB were in close agreement with previous biochemical data. A T663G/C24W polymorphic change in E7 correlated with a decrease in E7-pRb relative binding affinity the significance of which remains to be clarified. This semi-quantitative biochemical and genetic approach may be useful as a first step in the development of clinical protocols for the screening and identification of important HPV variants for clinical interpretation and for further functional analysis by transfection or other bioassays.
Assuntos
Variação Genética , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/metabolismo , Proteínas Repressoras , Proteína do Retinoblastoma/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Feminino , Humanos , Proteínas Oncogênicas Virais/genética , Teste de Papanicolaou , Papillomaviridae/classificação , Papillomaviridae/genética , Proteínas E7 de Papillomavirus , Infecções por Papillomavirus/virologia , Proteína do Retinoblastoma/genética , Análise de Sequência de DNA , Infecções Tumorais por Vírus/virologia , Esfregaço VaginalRESUMO
As a first step toward elucidation of the action of factors secreted by the epithelium of oviduct, differential display reverse transcription-polymerase chain reaction (DDRT-PCR) was used in this study to identify transcripts of such oviductal factors in gilts carrying various stages of early embryo development post hormone-induced ovulation. A total of 13 differentially expressed transcripts were identified between 50 and 120 hr post-hCG injection (between 1- and 8-cell embryonic stages). Twelve of these transcripts were found to be initially expressed at 96 hr post-hCG injection (at 4-cell embryonic stage) and beyond. Three of such genes were shown by sequence analysis to be the porcine transforming growth factor-alpha, the porcine transforming growth factor-beta-binding protein II and a porcine astral natriuretic factor receptor-like transcript. Only one differentially expressed gene was detected between 50-60 and 85 hr post-hCG injection, and this gene turned out to be the porcine follicle-stimulating hormone receptor. The remaining eight transcripts detected by DDRT-PCR were novel. Moreover, most of these newly expressed genes were found to be turned on at a time coincidental with that of the 4-cell block of porcine embryos cultured in vitro. Our results demonstrate that DDRT-PCR is a feasible approach for rapid identification of genes that are differentially expressed in oviductal epithelium. Some of the genes thus identified may be important for unhindered development of embryos in the oviduct.
Assuntos
Tubas Uterinas/embriologia , Receptores do FSH/genética , Fator de Crescimento Transformador alfa/genética , Animais , Epitélio , Feminino , Expressão Gênica , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SuínosRESUMO
A simplified version of a PCR-based reductional restriction fragment length polymorphism (rRFLP) approach for typing of human papillomaviruses (HPVs) is described previously [Wang et al., 1997]. It is achieved by the use of a biotin-labeled primer in PCR which, on restriction digestion and staining, is associated with only a single restriction fragment. In this report, we describe a further development of the rRFLP approach with the use of a fluorescence-labeled primer in PCR and fragment detection by laser scanning in an automatic sequencer. HPV typing is achieved by computer-assisted matching of the fluorescence-labeled rRFLP patterns with a database of rRFLP patterns of all known anogenital HPV types. On analysis of the typing of 133 HPV-positive cases using this procedure, 20 different HPV types were detected in exfoliated cervical cells in PAP smear samples derived from Taiwanese women. The results indicate the existence of a heterogeneous population of HPV types in Taiwan. Although most cases were associated with the more common HPV types, a significant fraction (about 20%) of the HPV types detected was related to the less common genotypes, which are often not included in commercial kits available for HPV typing. The results indicate the importance of covering as many HPV types as possible in clinical HPV genotyping protocols.
Assuntos
Papillomaviridae/classificação , Polimorfismo de Fragmento de Restrição , Colo do Útero/virologia , Primers do DNA , Feminino , Corantes Fluorescentes , Humanos , Papillomaviridae/genética , Infecções por Papillomavirus/virologia , Reação em Cadeia da Polimerase/métodos , Software , Infecções Tumorais por Vírus/virologiaRESUMO
Familial dysalbuminemic hyperthyroxinemia (FDH) is an autosomal dominant disorder characterized by euthyroid hyperthyroxinemia. However, FDH has not been reported in Chinese or African patients. Here, we report the first case of FDH in a Chinese patient. A 69-year-old Chinese man was found to have increased serum total T(4) concentrations (198-242nmol/l; normal range 58-148nmol/l) and free T(4) concentrations (>58pmol/l; T(4) analog method, normal range 9-28pmol/l). Serum total T(3) and TSH concentrations were normal. The patient was misdiagnosed as hyperthyroid and was later suspected to have a TSH-producing tumor by the finding of a pituitary microadenoma, which was eventually proven to be a non-functional pituitary 'incidentaloma'. Electrophoretic analysis of the patient's serum proteins demonstrated enhanced albumin binding of [(125)I]T(4). Serum free T(4) concentrations were normal (16-19pmol/l, normal range 9-26pmol/l) when a two-step method was used. Direct sequencing of the albumin gene showed a guanine to adenosine transition in the second nucleotide of codon 218, resulting in a substitution of histidine (CAC) for the normal arginine (CGC) in one of the two alleles in the patient. The point mutation was further confirmed by HphI digestion of exon 7 of the albumin gene. The patient's son was not affected. Our studies demonstrated that the point mutation of the albumin gene in a Chinese patient with FDH was similar to that found in western white families, but differed from that in a Japanese family in whom a guanine to cytosine transition at the same position was found.
Assuntos
Povo Asiático/genética , Genes Dominantes , Hipertireoxinemia/genética , Mutação Puntual , Albumina Sérica/genética , Idoso , Substituição de Aminoácidos , Arginina/química , Códon , Histidina/química , Humanos , Masculino , TaiwanRESUMO
A sensitive technique for the sexing of bovine embryos was developed using polymerase chain reaction (PCR) amplification of the bovine amelogenin (bAML) gene on the X- and Y-chromosomes of Holstein dairy cattle. Cloning and DNA sequencing showed a 45.1% homology between the fifth intron of the bAML-X and bAML-Y gene with multiple deletions. A pair of sex-specific primers was designed to allow amplification of a single fragment of 467-bp from the X-chromosome of female cattle and two fragments of 467-bp and 341-bp from the X- and Y-chromosomes of male cattle. The primers were successfully applied to bovine sexing from single blastomeres isolated from day-6 to day-7 cow embryos by direct cell lysis and PCR. Our protocol of embryo sexing should be applicable to the diagnosis of defective genes in vitro in human embryos and in other domestic or recreational animals.
Assuntos
Blastômeros , Proteínas do Esmalte Dentário/genética , Reação em Cadeia da Polimerase/métodos , Processos de Determinação Sexual , Cromossomo X/genética , Cromossomo Y/genética , Amelogenina , Animais , Sequência de Bases , Southern Blotting , Bovinos , Clonagem Molecular , Feminino , Amplificação de Genes , Íntrons , Masculino , Modelos Genéticos , Dados de Sequência Molecular , Polimorfismo Genético/genética , Sensibilidade e Especificidade , Homologia de Sequência do Ácido NucleicoRESUMO
OBJECTIVE: To identify the site of fetal blood sampling (FBS) with lesser complications; and also analyses the reasons for targetting the intrahepatic vein (IHV) for FBS. METHODS: Fetal blood sampling (FBS) performed on 382 women over a period of 7 years at the National University Hospital, Singapore was analysed. FBS was performed from 13 weeks of gestational age onwards. In 76.4% (292 of 382) the intrahepatic part of the umbilical vein (IHV) was targetted; in 18.3% (70 of 382) percutaneous umbilical cord sampling (PUBS) was performed; in 5.2% (20 of 382) cardiocentesis was performed to obtain fetal blood. RESULTS: Multivariate analysis showed an increase in odds of fetal loss for umbilical cord and cardiocentesis groups compared with the IHV FBS group. It was statistically significant (p < 0.01) only in the cardiocentesis group for fetal loss at < 2 weeks of performing the procedure.
Assuntos
Sangue Fetal/fisiologia , Veias Hepáticas/embriologia , Diagnóstico Pré-Natal/métodos , Feminino , Morte Fetal/etiologia , Idade Gestacional , Veias Hepáticas/fisiologia , Humanos , Idade Materna , Análise Multivariada , Razão de Chances , Gravidez , Resultado da Gravidez , Diagnóstico Pré-Natal/efeitos adversos , Análise de Regressão , Estudos Retrospectivos , Ultrassonografia Pré-Natal , Veias Umbilicais/embriologia , Veias Umbilicais/fisiologiaRESUMO
With the advancement of techniques in molecular biology, rapid, sensitive, and reliable methods of DNA typing for parentage testing have become available. In this study, we evaluated the usefulness of multiplex polymerase chain reaction (PCR) with 12 unlinked short tandem repeat (STR) loci for paternity testing in Taiwan. The genetic informativeness of this test was then compared with that of conventional human leukocyte antigen (HLA) analysis in 167 parentage studies. The 12 STR loci alone provided a cumulative power of exclusion of up to 0.9998. Paternity was excluded in 59 (35.3%) cases, including 40 of 112 paternity trios and 19 of 55 paternity duos. In the 40 trios in which paternity was excluded, a mean of 6 (range, 3-9) incompatible STR markers were in the 19 duos in which paternity was excluded, a mean of 4 (range, 1-8) incompatible STR markers were noted. In the 72 trios in which the alleged paternity could not be excluded, the mean probabilities of paternity (PP) were 90.6863% with HLA testing alone, 99.9847% with STR analysis alone, and 99.9972% with combined HLA and STR analysis. In the 36 duos in which the alleged paternity could not be excluded, the mean PPs were 81.4768% with HLA testing alone, 99.6124% with STR analysis alone, and 99.9145% with combined HLA and STR analysis. These results suggest that STR analysis is very powerful when used alone for paternity trio testing and when combined with conventional serologic HLA typing for duo parentage testing in the Taiwan population.
Assuntos
Mapeamento Cromossômico , Paternidade , Sequências de Repetição em Tandem , Feminino , Teste de Histocompatibilidade , Humanos , Masculino , Reação em Cadeia da Polimerase , Polimorfismo GenéticoRESUMO
Synthetic antisense oligodeoxynucleotides (ODNs) and a system containing transcription and translation coupled rabbit reticulocyte lysate were used to develop a new model modulating the synthesis of small delta antigen which, in turn, inhibits the replication of HDV (hepatitis D virus). The ODN was stable for at least 50 min in this system at 37 degrees C. Unmodified 15-mer antisense D3 and D4, complementary to translation initiation region and coding region, respectively, inhibit the synthesis of small delta antigen by 95% at a concentration of 5 microM, whereas antisenses complementary to 5' noncoding region, stop codon region and polyadenylation site were less effective. This system also showed a dose-dependent inhibitory effect of antisense D3 on the production of the target protein. However, the synthesis of E6 protein, an internal control, was not affected. These observations imply that this in vitro system is convenient for rapid screening of effective antisense compounds and offers a promising perspective for the investigation of translation mechanisms and for the inhibition of HDV replication by antisense strategy.
Assuntos
Antígenos de Hepatite/efeitos dos fármacos , Antígenos de Hepatite/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Animais , Sistema Livre de Células , Vírus Defeituosos , Relação Dose-Resposta a Droga , Estabilidade de Medicamentos , Vírus Delta da Hepatite/efeitos dos fármacos , Vírus Delta da Hepatite/genética , Vírus Delta da Hepatite/metabolismo , Antígenos da Hepatite delta , Oligonucleotídeos Antissenso/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Coelhos , Reticulócitos/química , Reticulócitos/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
The proliferation and differentiation of hematopoietic cells are stimulated by a group of glycoproteins called colony stimulating factors (CSFs). Previously, we found that the human hepatoma cell line HA22T/VGH secreted a high level of human granulocyte-macrophage colony-stimulating factor (hGM-CSF). The cDNA of hGM-CSF, including the signal peptide sequence, was amplified from the total RNA of HA22T/VGH by a reverse-transcription polymerase chain reaction and was cloned into the pUC18 vector. After confirming the nucleotide sequence, the cDNA was inserted into a pVL1393 baculovirus transfer vector. The recombinant baculovirus carrying hGM-CSF cDNA was generated by co-transfecting the hGM-CSF recombinant transfer vector and BaculoGold baculovirus DNA into the Sf9 insect cells. The expected hGM-CSF transcript was detected in the recombinant virus-infected Sf9 cells. The conditioned media of the infected cells were analyzed by a slot-blot immunoassay. The results indicate that the infected insect cells produced and secreted hGM-CSF. According to colony forming assay, a maximum titer of 2.1 x 10(6). U ml-1 of hGM-CSF in the medium was obtained on the third day after infection.
Assuntos
Baculoviridae/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Biotecnologia , Medula Óssea/efeitos dos fármacos , Células da Medula Óssea , Linhagem Celular , Primers do DNA/genética , DNA Complementar/genética , Expressão Gênica , Vetores Genéticos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SpodopteraRESUMO
To discern the structural features of cellular loci that are disrupted by type 16 human papillomavirus (HPV-16) integration in cervical cancer, a polymerase chain reaction (PCR)-based strategy was employed for direct amplification and sequence analysis of four such cellular loci in cancer biopsy samples. One of the HPV-16-disrupted loci was found to be the microtubule-associated protein (MAP-2) gene and the other three loci were uncharacterized and were designated PID-1 to -3 (for papillomavirus integration-disrupted). The junctional sequences of the viral integration sites in the four loci analyzed are bracketed by long tracts of homogeneous purine or pyrimidine or alternating purine-pyrimidine which are known to destabilize the B-form conformation of the DNA structure. Using a panel of human/hamster hybrid cell DNAs and PCR analysis, the four loci were assigned to chromosomes 2 (MAP-2), 9 (PID-1), 1 (PID-2) and 8 (PID-3), respectively. These chromosomes carry numerous other previously determined viral integration and chromosomal fragile sites and the myc oncogenes. The PID-1 locus was further found in Southern analysis to be rearranged and amplified in another cervical cancer biopsy and a cervical carcinoma cell line (CaSki). On Northern analysis, the PID-1 and -3 probes detected a 3.0- and a 3.6-kb transcript, respectively, in normal cervical cells and in cervical cancer cell lines. The findings suggest that HPV-16 genome integrates frequently into topologically destabilized and transcriptionally active chromosomal sites. It remains to be elucidated whether the MAP-2 and the PID loci contribute to the pathogenesis of cervical cancer.
Assuntos
Papillomaviridae/genética , Infecções por Papillomavirus/virologia , Infecções Tumorais por Vírus/virologia , Neoplasias do Colo do Útero/virologia , Integração Viral , Sequência de Bases , Northern Blotting , Southern Blotting , Mapeamento Cromossômico , DNA Viral , Feminino , Humanos , Dados de Sequência Molecular , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/patologia , Células Tumorais Cultivadas , Infecções Tumorais por Vírus/patologia , Neoplasias do Colo do Útero/patologiaRESUMO
The majority of congenital adrenal hyperplasia (CAH) cases arise from mutations in the steroid 21-hydroxylase (CYP21) gene. Without reliance on HLA gene linkage analysis, we have developed primers for differential polymerase chain reaction (PCR) amplification of the CYP21 gene and the non-functional CYP21P gene. Using the amplification created restriction site (ACRS) approach for direct mutational detection, a secondary PCR was then performed using a panel of primers specific for each of the 11 known mutations associated with CAH. Subsequent restriction analysis allowed not only the detection but also the determination of the zygosity of the mutations analysed. Existing deletion of the CYP21 gene could also be detected. In the analysis of 20 independent chromosomes in 11 families of CAH patients in Taiwan, four CYP21 mutation types, besides deletion, were detected. Interestingly, in five different alleles, the CYP21P pseudogene contained some polymorphisms generally associated with the CYP21 gene. These results suggest gene conversion events that are occurring in both CYP21P and CYP21 genes. Our combined differential PCR-ACRS protocol is simple and direct and is applicable for prenatal diagnosis of CAH using chorionic villi or amniotic cells.
Assuntos
Hiperplasia Suprarrenal Congênita/enzimologia , Mutação , Esteroide 21-Hidroxilase/genética , Hiperplasia Suprarrenal Congênita/genética , Sequência de Bases , Análise Mutacional de DNA , Primers do DNA , Humanos , Masculino , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
Cloning of DNA fragments with blunt ends or identical protruding ends results in colonies with the insertion sequence existing in two possible orientations. The orientation and the junctional sequence of the positive clones need to be established by means of restriction analysis and/or sequencing. Here, we proposed a rapid one-step method for the screening of clones not only with the desired orientation, but also with an intact junctional sequence. In this method, a 16-18 meric oligonucleotide probe synthesized according to the expected vector-insert junctional sequence is used as a probe in colony hybridization screening. Using this strategy, only recombinant clones which fulfill the above criteria will show positive signals in hybridization.
Assuntos
Clonagem Molecular/métodos , DNA Recombinante/análise , Antígenos Nucleares do Vírus Epstein-Barr/biossíntese , Herpesvirus Humano 4/genética , Sondas de Oligonucleotídeos , Sequência de Bases , Primers do DNA , Antígenos Nucleares do Vírus Epstein-Barr/genética , Escherichia coli , Genes Virais , Vetores Genéticos , Herpesvirus Humano 4/metabolismo , Mutagênese Insercional , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase/métodosRESUMO
A transcription and translation coupled reticulocyte lysate system was established for rapid screening of antisense oligodeoxyribonucleotides (ODNs) to determine which are most effective for mRNA translation-arrest. A plasmid containing the target cDNA under the control of the T7 (or SP6) promoter was added to the lysate system in the presence of the T7 (or SP6) RNA polymerase, RNase H, and the antisense ODN under test. Transcription and translation were accomplished in a one-tube reaction. Translation-arrest caused by antisense ODN was evaluated in terms of the amounts of de novo-synthesized, [35S]-methionine or [35S]cysteine labeled target protein measured by gel electrophoresis and autoradiography. The properties of this system and optimal reaction conditions for use in antisense ODN screening were determined. Our method is simpler and more rapid than other in vitro screening methods.
Assuntos
Oligonucleotídeos Antissenso/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/genética , Sequência de Bases , Dados de Sequência MolecularRESUMO
To study the prevalence and type of the hepatitis B e antigen (HBeAg)-negative mutant in hepatitis D virus (HDV) superinfection, the precore region of hepatitis B virus (HBV) was analyzed by cycle sequencing. Of the 58 samples sequenced, 24 were wild type and 34 carried mutants. The precore stop mutation (TAG) at the 28th codon was found in 32 cases, other mutations were found in 7, and double mutations were found in 5. The absence of HBeAg showed a substantial agreement with the presence of mutants (kappa value, 0.74). Of the acute hepatitis patients, HDV replication and clinical manifestations were not significantly different between those with mutant and wild type virus, except that those with mutant virus were older (mean age, 48 vs. 28 years; P < .002). The absence of HBeAg in these patients is mainly due to HDV superinfection in older HBV carriers who already had precore mutant.
Assuntos
Antígenos do Núcleo do Vírus da Hepatite B/análise , Vírus da Hepatite B/genética , Hepatite B/virologia , Hepatite D/virologia , Vírus Delta da Hepatite/genética , Mutação , Superinfecção/virologia , Doença Aguda , Adulto , Sequência de Bases , Doença Crônica , DNA Viral/análise , Antígenos de Hepatite/análise , Hepatite B/imunologia , Hepatite B/fisiopatologia , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos E da Hepatite B/genética , Vírus da Hepatite B/imunologia , Hepatite D/imunologia , Hepatite D/fisiopatologia , Vírus Delta da Hepatite/imunologia , Antígenos da Hepatite delta , Humanos , Fígado/virologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Prevalência , RNA Viral/análise , Superinfecção/imunologia , Superinfecção/fisiopatologiaRESUMO
A panel of two poorly differentiated (HA22T/VGH and SK-Hep-1) and six well-differentiated (HuH-6-cl 5, HuH-7, PLC/PRF/5, Hep G2, Hep 3B, and Tong) human hepatocellular carcinoma (HCC) cell lines were studied for the production of colony-stimulating factors (CSFs) using the granulocyte and macrophage colony formation (CFU-GM) assay, immunocytochemical staining, and Northern blotting. Medium conditioned by untreated HA22T/VGH cells contained a high level of CSFs that could stimulate the in vitro colony formation of human myeloid progenitor cells. The HA22T/VGH cell-derived CSF had an apparent molecular weight of 23 kD. Its activity could be effectively neutralized by antiserum against granulocyte-macrophage CSF (GM-CSF) but not by antibodies to other hematopoietic growth factors, including G-CSF, M-CSF, interleukin-3 (IL-3), and IL-6. Correspondingly, immunocytochemical studies using monoclonal anti-GM-CSF showed a strong positive reaction in the cytoplasm of the HA22T/VGH cells. Northern blot analysis revealed that untreated HA22T/VGH cells expressed a considerable amount of GM-CSF mRNA, confirming that GM-CSF production was constitutive. At optimal concentrations, lipopolysaccharide (LPS), IL-1beta, interferon-gamma (IFN-gamma), and tumor-promoting phorbol diester (TPA) could all stimulate HA22T/VGH cells to secrete GM-CSF. In addition to HA22T/VGH, SK-Hep-1 cells could also produce GM-CSF, although less effectively, whereas all the well-differentiated HCC cell lines tested were negative for CSF production. Morphologic, cytochemical, and immunocytochemical examinations demonstrated that both poorly differentiated CSF-producing HCC cell lines (HA22T/VGH and SK-Hep-1) were macrophage-like in morphology, possessed nonspecific esterase (NSE) activity, and expressed CD14, CD68, and HLA-DR on their surface, while all the well-differentiated HCC cell lines were epithelioid and lacked myeloid differentiation antigens. These results suggest that monocytoid features and CSF production may be differentiation markers of hepatocytes at the immature stages, amd that the HA22T/VGH and SK-Hep-1 cell lines may be valuable tools for the study of hepatic function and differentiation.