Urinary cell-free DNA as a potential tumor marker for bladder cancer

The objective was to assess the possibility of measuring urine creatinine (UCr)-adjusted urinary cell-free (ucf) DNA concentration as a noninvasive screening tool for bladder cancer. Using PicoGreen-based detection, the ucf-DNA/UCr concentration was quantified in urine supernatant specimens from 46 bladder cancer patients and 98 controls and compared to 400-bp real-time PCR-based detection, which detected the amplification of 400-bp beta-actin (named 400-bp ucf-DNA/UCr). The mean concentrations for both PicoGreen and 400-bp ucf-DNA (ng/mL)/UCr (mg/dL) were significantly higher in bladder cancer patients than in controls: 15.28 vs 6.68 (p<0.001, t-test) and 14.98 vs 1.07 (p<0.001), respectively. Among different stages and grades, no significant difference was found between these two methods. The areas under the ROC curves of PicoGreen and 400-bp ucf-DNA/UCr were 0.571 (95% confidence interval, 0.451-0.692) and 0.805 (95% confidence interval, 0.713-0.896), respectively. In 400-bp ucf-DNA/UCr, the best sensitivity and specificity were 86.1% and 72.0% at the cutoff value of 0.0645. These data indicated that 400-bp ucf-DNA/UCr is more reliable for bladder cancer detection than PicoGreen. In conclusion, our results suggest that ucf-DNA/UCr can be used as a potential tumor marker for bladder cancer, especially for detecting longer DNA fragments.     

New integration vector using a cellulase gene as a screening marker for Lactobacillus

The new integration vector for Lactobacillus, pJC4, was developed using the extracellular endoglucanase A gene (celA) of Clostridium thermocellum as a screening marker. pJC4 was transformed into four Lactobacillus species, Lb. johnsonii, Lb. gasseri, Lb. bulgaricus, and Lb. plantarum. In each species, the pJC4 integrants were easily and accurately detected by the appearance of a clear halo on a cellulase screening plate without any false transformants. Polymerase chain reaction and Southern hybridization indicated that all transformants with clear halos contained pJC4 in their chromosomal DNAs. The celA gene could be a useful screening marker for other lactic acid bacteria.     

5-Methylcytosine as a marker for the monitoring of DNA methylation

The extent of the DNA methylation of genomic DNA as well as the methylation pattern of many gene-regulatory areas are important aspects with regard to the state of genetic information, especially their expression. There is growing evidence that aberrant methylation is associated with many serious pathological consequences. As genetic research advances, many different approaches have been employed to determine the overall level of DNA methylation in a genome or to reveal the methylation state of particular nucleotide residues, starting from semiquantitative methods up to new and powerful techniques. In this paper, the currently employed techniques are reviewed both from the point of view of their relevance in genomic research and of their analytical application. The methods discussed include approaches based on chromatographic separation (thin-layer chromatography, high-performance liquid chromatography, affinity chromatography), separation in an electric field (capillary electrophoresis, gel electrophoresis in combination with methylation-sensitive restriction enzymes and/or specific sequencing protocols), and some other methodological procedures (mass spectrometry, methyl accepting capacity assay and immunoassays).     

Ribosomal repeat in cell free DNA as a marker for cell death

A novel method for in vivo evaluation of cell death in patients with acute and/or chronic heart diseases, accompanied by apoptosis or cell necrosis has been developed. The method is based on the analysis of cell free DNA (cfDNA) in the blood serum (plasma). It includes estimation of concentration of serum ribosomal repeat (rDNA), content of rDNA in total cfDNA, as well as factors of cfDNA elimination, such as nuclease activity and anti-DNA antibody. We have found a fivefold increase in serum cfDNA concentration and a 12-fold increase of serum rDNA concentration in patients with acute myocardial infarction compared with healthy individuals. In chronic coronary ischemia serum cfDNA concentration was nearly normal, but the content of rDNA in cfDNA was 4.8-fold higher, and the serum rDNA concentration was increased sevenfold. We hypothesize that putative reason for accumulation of rDNA within cfDNA might be attributed to the previously reported resistance of rDNA to the ds-fragmentation by serum endonucleases. In acute and chronic coronary disease serum nuclease activity and the titer of anti-DNA antibodies (which are mainly bound to the cfDNA) was substantially higher than in the healthy controls. It is suggested that release of rDNA fragments into blood not only reflects cellular death in the body but also determines the response of the organism to the disease-associated stress.     

DNA methylation signatures in circulating cell-free DNA as biomarkers for the early detection of cancer

Detecting cell-free DNA(cfDNA) or circulating tumor DNA(ctDNA) in plasma or serum could serve as a "liquid biopsy", which would be useful for numerous diagnostic applications. cfDNA methylation detection is one of the most promising approaches for cancer risk assessment. Here, we reviewed the literature related to the use of serum or plasma circulating cell-free DNA for cancer diagnosis in the early stage and their power as future biomarkers.     

Basta tolerance as a selectable and screening marker for transgenic plants of Norway spruce

The bar gene conferring resistance to the herbicide Basta (containing phosphinothricin) was transferred to embryogenic cultures of Picea abies by particle bombardment and transformants were selected on Basta medium. In total, 83 9-month-old transgenic plants of Picea abies from six transformed sublines were analysed for continued tolerance to Basta. PCR analysis showed that the bar gene was present in all transformed plants but not in the control plants. Northern blot analysis showed differences in expression level among plants from the same subline as well as among sublines. A simple biotest for screening for Basta tolerance based on the colour change of detached needles induced by Basta was developed. The tolerance to Basta varied among the plants from different sublines. Needles from four of the sublines were resistant to 100 mg l⁻¹ phosphinothricin, a concentration inducing yellowing in control needles, while plants from the other two sublines were on average two to four times as resistant as untransformed control plants. The biotest enables rapid semi-quantitative monitoring for continued transgene expression in long-lived tree species. Received: 21 October 1999 / Revision received: 24 January 2000 / Accepted: 24 January 2000     

Random amplified polymorphic DNA fingerprinting as a marker for Paramecium jenningsi strains

The aim of the present study is to establish a common RAPD marker for P. jenningsi using a series of Ro primers and to investigate if strains originating from distant and isolated localities (Japan, China, India, Saudi Arabia) have isolated gene pools and represent distinct species. An analysis of dendrograms constructed on the basis of RAPD-PCR fingerprints with four primers (Ro 460-04, 460-06, 460-07, and 460-10) from the first part of this project (SKOTARCZAK et al. 2004), assigns the strains to two groups consisting of the continental strains (India, Saudi Arabia, China) and Japanese strains that have been considered as a separate sibling species within P. jenningsi. The genetic similarity of the Indian and Arabian strains was ascertained, whereas the Chinese strain formed an independent branch in this sibling species. The primers Ro (460-01,460-02, 460-03, 460-05, 460-08) also distinguish between two groups of strains, although they divide the Japanese strains into two subgroups that are not reproductively isolated. This probably indicates genetic variation within this sibling species. However, it comprises one common gene pool (successful inter-strain crosses) and is reproductively isolated from the other sibling species. The results presented in these papers confirm that the construction of ten band patterns having marker attributes is possible on the basis of DNA amplification from 9 strains of P. jenningsi with the RAPD-PCR fingerprinting method using five primers from the Ro series. The patterns can be assigned to three marker-groups: a general species group, a group differentiating between sibling species, and accessory strain markers.     

Filamin-A as a marker and target for DNA damage based cancer therapy

Filamin-A, also called actin binding protein 280 (ABP-280), cross-links the actin filaments into dynamic orthogonal network to serve as scaffolds in multiple signaling pathways. It has been reported that filamin-A interacts with DNA damage response proteins BRCA1 and BRCA2. Defects of filamin-A impair the repair of DNA double strand breaks (DSBs), resulting in sensitization of cells to ionizing radiation. In this study, we sought to test the hypothesis that filamin-A can be used as a target for cancer chemotherapy and as a biomarker to predict cancer response to therapeutic DNA damage. We found that reduction of filamin-A sensitizes cancer cells to chemotherapy reagents bleomycin and cisplatin, delays the repair of not only DSBs but also single strand breaks (SSBs) and interstrand crosslinks (ICLs), and increases chromosome breaks after the drug treatment. By treating a panel of human melanoma cell lines with variable filamin-A expression, we observed a correlation between expression level of filamin-A protein and drug IC(50). We further inhibited the expression of filamin-A in melanoma cells, and found that this confers an increased sensitivity to bleomycin and cisplatin treatment in a mouse xenograft tumor model. These results suggest that filamin-A plays a role in repair of a variety of DNA damage, that lack of filamin-A is a prognostic marker for a better outcome after DNA damage based treatment, and filamin-A can be inhibited to sensitize filamin-A positive cancer cells to therapeutic DNA damage. Thus filamin-A can be used as a biomarker and a target for DNA damage based cancer therapy.     

Potential of carotenoids in aquatic yeasts as a phylogenetically reliable marker and natural colorant for aquaculture

Apart from Xanthophyllomyces dendrorhous, pink colony-forming yeasts have not been examined as a pigmentation source in captive animals. In this study, aquatic yeasts were screened with a view to abundances of carotenoids. Phylogenetic analyses of these caroetnoid-rich yeasts based on large subunit ribosomal RNA gene (LSU rDNA) partial sequences showed that all belonged to the order Sporidiobolales. Both the qualitative and the quantitative differences in carotenoids between the yeasts appeared to be consistent with their phylogenetic affiliations. This information might be useful in the selection of pigment-rich yeasts containing specific carotenoids from a large number of strains. We also found, for the first time, the potential of a pigment-rich Rhodotorula strain as a colorant for aquaculture. The integuments of tilapia and carp fed the alkali-treated cells of strain Rhodotorula dairenensis Sag 17 were pigmented after 3 months of cultivation. The fish integuments retained the yeast carotenes shortly after the start of feeding, and were converted to the fish-specific xanthophylls in vivo.     

Mitochondrial DNA as a marker of molecular diversity: a reappraisal

Over the last three decades, mitochondrial DNA has been the most popular marker of molecular diversity, for a combination of technical ease-of-use considerations, and supposed biological and evolutionary properties of clonality, near-neutrality and clock-like nature of its substitution rate. Reviewing recent literature on the subject, we argue that mitochondrial DNA is not always clonal, far from neutrally evolving and certainly not clock-like, questioning its relevance as a witness of recent species and population history. We critically evaluate the usage of mitochondrial DNA for species delineation and identification. Finally, we note the great potential of accumulating mtDNA data for evolutionary and functional analysis of the mitochondrial genome.     

Potential for cell culture techniques as a wildlife management tool for screening primary repellents

The identification of new chemical repellents for wildlife damage management is impeded by the need to perform cumbersome and expensive behavioral tests. Here, we report the development of in vitro cell culture methods to increase the speed and efficiency of repellent screening while reducing costs, as well as the number of animals necessary for research. Our methods exploit the fact that effective primary repellents depend on the stimulation of pain receptors. We cultured trigeminal nociceptors (pain receptors) from Norway rat (Rattus norvegicus, laboratory strain), white leghorn chicken (Gallus gallus), coyote (Canis latrans), white-tailed deer (Odocoileus virginianus) and Canada goose (Branta canadensis), and then applied digital fluorescence microscopy to measure changes in intracellular calcium (an index of cellular activation) in response to applications of known and effective repellents. Capsaicin, bradykinin and acetylcholine were more effective stimuli for rat, coyote, and deer neurons than cells from chicken.     

Fast screening target sites for RNA interference using a cell-free system

A fast and simple procedure to screen target sites for RNA interference by using RNA in a cell-free system of Hela cells, and then evaluating the efficiency by Northern blotting, is described. This procedure produces results with an identical reliability compared to those described previously but which are more time-consuming than this present method.     

Effects of chloramphenicol on plant cells: Potential as a selectable marker for transformation studies

The growth of tobacco and carrot cells in suspension cultures and on solidified medium was strongly inhibited by the antibiotic chloramphenicol. This effect was irreversible. The drug had no effect on [³H]-uridine incorporation but inhibited protein synthesis and induced striking morphological alterations in intoxicated cells. O₂ consumption was also reduced in treated cells. Tobacco and carrot cells were unable to inactivate chloramphenicol by enzymatic acetylation.     

Application of retrovirus-mediated expression cloning for receptor screening of a parasite

Retrovirus-mediated expression cloning has been applied in both virology and cell biology. Although there is some difficulty in applying this technique to screening for a receptor recognized by an intracellular parasite, we modified the conventional method to identify a putative receptor for the Plasmodium falciparum BAEBL protein. We show that this method is effective in screening for a parasite receptor.     

M13 phage DNA as a universal marker for DNA fingerprinting of animals, plants and microorganisms

Hypervariable polymorphic patterns were detected with M13 phage DNA as a probe in genomic DNA of organisms belonging to different taxonomic groups including animals (vertebrates and invertebrates), plants and microorganisms. Individual-specific restriction pattern analysis (DNA fingerprinting) with this probe proved to be useful for individual identification, analysis of somatic stability and paternity testing in man. The nuclear type of inheritance indicates that the hypervariable DNA regions in question are located in the chromosomes, not in the mitochondrial DNA. The data obtained also demonstrate a potential range of M13 DNA applications as a probe for DNA fingerprinting of animals, plants and microorganisms, particularly for the determination of inbred lines, identification of bacterial strains and establishing stock, variety and strain distinctions.