Cervical cancer is the second most prevalent malignancy among women worldwide, and additional objective diagnostic markers for this disease are needed. Given the link between cancer development and alternative splicing, we aimed to analyze the splicing patterns of the PRDX2, RAB1A, RAB1B, RAB5A and RAB25 genes, which are associated with different cancers, in normal cervical tissue, preinvasive cervical lesions and invasive cervical tumors, to identify new objective diagnostic markers. Biopsies of normal cervical tissue, preinvasive cervical lesions and invasive cervical tumors, were subjected to rapid amplification of cDNA 3' ends (3' RACE) RT?PCR. Resulting PCR products were analyzed on agarose gels, gel?purified and sequenced. Normal cervical tissue, preinvasive cervical lesions and invasive cervical tumors contained one PCR product corresponding to full?length PRDX2, RAB5A and RAB25 transcripts. All tissues contained two RAB1A?specific PCR products corresponding to the full?length transcript and one new alternatively spliced RAB1A transcript. Invasive cervical tumors contained one PCR product corresponding to the full?length RAB1B transcript, while all normal cervical tissue and preinvasive cervical lesions contained both the full?length RAB1B transcript and three new alternatively spliced RAB1B transcripts. Alternative splicing of the RAB1A transcript occurs in all cervical tissues, while alternative splicing of the RAB1B transcript occurs in normal cervical tissue and in preinvasive cervical lesions; not in invasive cervical tumors.
The aim of this study was to test if an extremely weak 1 GHz electromagnetic field (EMF), known to be in resonance with clusters of water molecules, has biological effects on human fibroblasts. We demonstrated that in an in vitro model of wound healing, this EMF can activate fibroblast migration. [(3)H]thymidine incorporation experiments demonstrated that the EMF could also activate fibroblast proliferation. Activation of the expression of human fibroblast growth factor 1 (HFGF1) after EMF exposure showed that molecular wound healing pathways are activated in response to this water-resonant EMF.
Increased production of reactive oxygen species (ROS) in mitochondria has been proposed as the pathogenic mechanism for chronic complications of diabetes. Mitochondrial DNA (mtDNA) is more vulnerable to reactive oxygen species. However, there are few data on the mitochondrial DNA damage in diabetes and these are available only from patients with different duration of the disease and tissues not relevant to the chronic complications of diabetes. We therefore proposed to study the stability of mitochondrial DNA under controlled experimental conditions, to understand its contribution to chronic complications of diabetes.
We have investigated promoter methylation of the Insr, Igf1 and Igf1r genes in skeletal and cardiac muscles of normal and diabetic db/db mice. No differences in Insr promoter methylation were found in the heart and skeletal muscles and no methylation was detected in the Igf1 promoter in skeletal muscle. In skeletal muscle, db/db males exhibited a 7.4-fold increase in Igf1r promoter methylation, which was accompanied by a 1.8-fold decrease in Igf1r mRNA levels, compared with controls. More than 50% of the detected methylation events were concentrated within an 18 bp sequence that includes one of the Sp1 binding sites. We conclude that the methylation level and pattern of the Igf1r promoter in skeletal muscle is related to gender and the diabetic state.
In the fields of proteomics, metabolic engineering and synthetic biology there is a need for high-throughput and reliable cloning methods to facilitate construction of expression vectors and genetic pathways. Here, we describe a new approach for solid-phase cloning in which both the vector and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable for efficient subcloning in an automated manner to rapidly generate a large number of gene constructs in various vectors intended for high throughput applications.
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