RESUMO
Cadmium (Cd), an economically valuable metal, is widely used in various industrial processes. Although it is of economic value, it is hazardous to human health. Cd accumulates in vital organs where it causes various diseases. Natural compounds with chelating or antioxidant properties have been tested to reduce the toxic effect of Cd. The antioxidant, antidiabetic and hypocholesterolemic properties of fenugreek (Trigonella foenum-graecum) leaves make it a candidate for investigation as protective agent against Cdinduced toxicity. In the present study, the protective effects of fenugreek leaf extract (FLE) on cell viability, morphology, and whole genomic transcription in cadmium chloride (CdCl2)treated rat liver cells were analyzed. The cells were treated with 25 µM CdCl2 alone, or cotreated with 5 µg/ml FLE for 48 h. The cotreated cells were pretreated with FLE for 2 or 4 h, followed by CdCl2 treatment. Genomic transcription analysis was performed in the CdCl2treated cells following treatment for 6 h. The CdCl2 caused a significant decrease in viability (35.8±4.1%) and morphological distortion of the cells, compared with the untreated control cells; whereas 4 h pretreatment with FLE (5 µg/ml) reversed the Cdinduced morphology alteration and increased the cell viability to 102±3.8%. Genomic transcription analysis of the CdCl2 onlytreated cells showed 61 upregulated and 124 downregulated genes, compared with 180 upregulated and 162 downregulated genes in the FLE pretreated cells. Furthermore, 37 and 26% of the affected total genomic genes in the CdCl2 onlytreated cells were involved in binding and catalytic activities, respectively, whereas 50 and 20% of the genes in the FLE pretreated cells were involved in binding and catalytic activities, respectively. In conclusion, these results suggested that genome transcriptome modulation may be important in the protective effect of FLE against Cdinduced toxicity in normal rat liver cells.
Assuntos
Cádmio/efeitos adversos , Hepatócitos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Substâncias Protetoras/farmacologia , Transcriptoma/efeitos dos fármacos , Trigonella/química , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Extratos Vegetais/química , Folhas de Planta/química , RatosRESUMO
Variation in genome structure is an important source of human genetic polymorphism: It affects a large proportion of the genome and has a variety of phenotypic consequences relevant to health and disease. In spite of this, human genome structure variation is incompletely characterized due to a lack of approaches for discovering a broad range of structural variants in a global, comprehensive fashion. We addressed this gap with Optical Mapping, a high-throughput, high-resolution single-molecule system for studying genome structure. We used Optical Mapping to create genome-wide restriction maps of a complete hydatidiform mole and three lymphoblast-derived cell lines, and we validated the approach by demonstrating a strong concordance with existing methods. We also describe thousands of new variants with sizes ranging from kb to Mb.
Assuntos
Genoma Humano , Mapeamento por Restrição Óptica/métodos , Algoritmos , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Variação Genética , Estudo de Associação Genômica Ampla , Humanos , Mola Hidatiforme/genética , Linfócitos/metabolismo , Mapeamento por Restrição Óptica/estatística & dados numéricos , Gravidez , Neoplasias Uterinas/genéticaRESUMO
The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos. Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF). Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days. The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state. Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.
Assuntos
Técnicas Citológicas/métodos , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Células Germinativas/citologia , Animais , Feminino , Gônadas/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Óvulo/citologiaRESUMO
Tumors lacking DNA mismatch repair activity (MMR) from patients with Hereditary Nonpolyposis Colorectal Cancer (HNPCC) or those with sporadic colorectal cancer can be identified by the presence of high levels of instability in repetitive sequences known as microsatellites (MSI). The assessment of MSI phenotype in human tumors helps to establish a clinical diagnosis and is accomplished with a reference panel of five mononucleotide repeats. By contrast, detection of MSI in mouse tumors has proven to be problematic and lack of a uniform set of markers for classification of MSI has impeded comparison of results between studies. We tested for MSI in intestinal tumors from MMR-deficient mice with four mononucleotide repeats with polyA(24-37) tracts and three new markers with extended polyA(59-67) tracts. All seven markers were sensitive to MSI in MMR-deficient tumors, but those with extended mononucleotide tracts displayed larger deletions, which were easily distinguishable from the germline alleles. With a panel of the five most sensitive and specific mononucleotide repeats, a high level of MSI was detected in 100% of MMR-deficient tumors, but not in tumors with MMR activity. This novel panel is an improvement over existing combinations of mono- and dinucleotide repeat markers and should facilitate MSI screening and standardize results from different studies.
Assuntos
Pareamento Incorreto de Bases/fisiologia , Instabilidade Genômica , Neoplasias Intestinais/diagnóstico , Neoplasias Intestinais/genética , Repetições de Microssatélites/genética , Sequências Repetitivas de Ácido Nucleico , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Genes APC/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologiaRESUMO
The aetiology of compromised spermatogenesis is often genetic in nature. There are only a few reports of father/son cohorts that have been evaluated to assess heritability of mutations associated with male factor infertility and the psychological well-being of the children. In the present study, multiple tissues were sampled from consenting male patients and their sons derived from intracytoplasmic sperm injection (ICSI) and underwent chromosomal and genetic analyses. Paediatric and psychological examinations were also conducted. In 87 men and 47 boys, 22 sequence tagged sites (STS) were analysed by multiplex PCR and deletion breakpoints were defined with additional loci. In one patient, the breakpoints map to the highly unstable palindrome-rich region within AZFb and proximal AZFc was investigated. A total of 86 blood, 26 semen, and 73 cheek cells samples were collected from adults, and 36 blood samples and 44 cheek cell specimens were obtained from the boys. Though all of the fathers had normal karyotypes, the incidence of chromosomal abnormalities in the somatic cells of male progeny was 8.3% (3/36). The incidence of germ line aneuploidy in these men was 0.5-2.8%. A CF mutation (Delta508) was detected in one of 87 men (1.2%) and microdeletions in Yq AZF were detected in 3.4% of 87 adults and in 2.1% of their sons (n = 47). In conclusion, screening for Y chromosome microdeletions provides crucial information in the counselling of couples seeking infertility treatment. Moreover, DNA extraction and Y deletion assessments of cheek cells provide a non-invasive approach. Inheritance of Yq deletions appears not to affect the psychological and physical development of children derived from ICSI.