The application of methylation specific electrophoresis (MSE) to DNA methylation analysis of the 5' CpG island of mucin in cancer cells

<p>Abstract</p> <p>Background</p> <p>Methylation of CpG sites in genomic DNA plays an important role in gene regulation and especially in gene silencing. We have reported mechanisms of epigenetic regulation for expression of mucins, which are markers of malignancy potential and early detection of human neoplasms. Epigenetic changes in promoter regions appear to be the first step in expression of mucins. Thus, detection of promoter methylation status is important for early diagnosis of cancer, monitoring of tumor behavior, and evaluating the response of tumors to targeted therapy. However, conventional analytical methods for DNA methylation require a large amount of DNA and have low sensitivity.</p> <p>Methods</p> <p>Here, we report a modified version of the bisulfite-DGGE (denaturing gradient gel electrophoresis) using a nested PCR approach. We designated this method as methylation specific electrophoresis (MSE). The MSE method is comprised of the following steps: (a) bisulfite treatment of genomic DNA, (b) amplification of the target DNA by a nested PCR approach and (c) applying to DGGE. To examine whether the MSE method is able to analyze DNA methylation of mucin genes in various samples, we apply it to DNA obtained from state cell lines, ethanol-fixed colonic crypts and human pancreatic juices.</p> <p>Result</p> <p>The MSE method greatly decreases the amount of input DNA. The lower detection limit for distinguishing different methylation status is < 0.1% and the detectable minimum amount of DNA is 20 pg, which can be obtained from only a few cells. We also show that MSE can be used for analysis of challenging samples such as human isolated colonic crypts or human pancreatic juices, from which only a small amount of DNA can be extracted.</p> <p>Conclusions</p> <p>The MSE method can provide a qualitative information of methylated sequence profile. The MSE method allows sensitive and specific analysis of the DNA methylation pattern of almost any block of multiple CpG sites. The MSE method can be applied to analysis of DNA methylation status in many different clinical samples, and this may facilitate identification of new risk markers.</p

Action spectra of apoptosis induction and reproductive cell death in L5178Y cells in the UV-B region

We investigated the action spectra for the induction of apoptosis and reproductive cell death in mouse lymphoma L5178Y cells using a high-performance spectroirradiator, the Okazaki Large Spectrograph at the National Institute for Basic Biology, Okazaki. L5178Y cells were exposed to monochromatic light at different wavelengths in the UV-B and UV-A regions. The frequencies of apoptosis induction and reproductive cell death were determined by counting cells with chromatin condensation and by a semi-solidified agarose colony formation assay, respectively. The measured action spectra for the two end-points were similar. The sensitivity decreased steeply with an increase of wavelength in the UV-B region, but showed no further decrement in the UV-A region. The action spectra were slightly steeper than that for the minimum erythematic dose (MED), and were similar to the light-absorption spectrum of DNA in the UV-B region. On the other hand, in the UV-A region, the spectra for both endpoints were close to the MED, but not to DNA absorption spectra. The difference between the measured spectra and that for MED may have been caused by the absorption of the light by the skin. Differences in the time course and morphological difference of apoptosis were found between the UV-B and UV-A region. These results suggest that although DNA damage induced by UV-B light can trigger apoptosis, or lead to reproductive cell death, other damage (membrane, protein and so on) may trigger those effects in the UV-A region

Distinct responses of chloroplasts to blue and green laser microbeam irradiations in the centric diatom Pleurosira laevis

The centric diatomPleurosira laevis is a large unicellular alga, in which ca 200 chloroplasts migrate toward the nuclear cytoplasm through the transvacuolar cytoplasmic strands in response to blue-light irradiation and, on the contrary, toward the cortical cytoplasm in response to green-light irradiation. We analyzed these light-induced chloroplast migrations using a scanning laser microbeam provided by a confocal microscope for intracellular irradiation. Spot irradiation of a blue laser microbeam induced rapid assemblage of chroloplasts into the nuclear cytoplasm regardless of the spot position and spot number. On the other hand, one or two spots of green laser microbeam induced chloroplast accumulation at the spots, although increasing spot numbers suppressed chloroplast accumulation at each spot. In our experimental condition, ca 1 min of blue-light irradiation was sufficient to stimulate movement, whereas green-light irradiation required uninterrupted and longer irradiation time (ca 15 min). Chloroplast assemblage induced by blue-light required extracellular Ca 2+, and was inhibited by Ca 2+channel antagonists. Furthermore, higher efficiencies of chloroplast migration were obtained when a single beam spot was fragmented and scattered over wider area of plasma membrane. These observations suggested that blue-light induced a response at the plasma membrane, which subsequently activated Ca 2+permeable channels. This sequence of physiological events is identical to what was previously observed with chloroplast movement in response to mechanical stimulation. Furthermore, experiments with the cytoskeleton-disrupting agents, colchicine and cytochalasin D, indicated that blue-light-induced chloroplast movement required microtubules whereas the green-light-induced response to beam spot required actin filaments

Equal-quantum action spectra indicate fluence-rate-selective action of multiple photoreceptors for photomovement of the thermophilic cyanobacterium Synechococcus elongatus

Unicellular thermophilic cyanobacterium Synechococcus elongatus displayed phototaxis on agar plate at 55°C. Equal-quantum action spectra for phototactic migration were determined at various fluence rates using the Okazaki Large Spectrograph as the light source. The shapes of the action spectra drastically changed depending on the fluence rate of the unilateral monochromatic irradiation: at a low fluence rate (3 μmol/m 2/s), only lights in the red region had significant effect; at a medium fluence rate (10 μmol/m 2/s), four major action peaks were observed at 530 nm (green), 570 nm (yellow), 640 nm (red) and 680 nm (red). At high fluence rates (30-90 μmol/m 2/s), the former two peaks remained, while red peaks at 640 nm and 680 nm disappeared and, interestingly, an action peak around 700-740 nm (far-red) newly appeared. These results indicate that two or more distinct photoreceptors are involved in the phototaxis and that suitable photoreceptors are selectively active in response to the stimulus of light fluence rates. Far-red or red background lights irradiated vertically from above drastically inhibited phototaxis toward red light or far-red light, respectively. These results indicate involvement of some phytochrome(s)

Blue and red light-induced germination of resting spores in the red-tide diatom Leptocylindrus danicus

Photophysiological and pharmacological approaches were used to examine light-induced germination of resting spores in the red-tide diatom Leptocylindrus danicus. The equal-quantum action spectrum for photogermination had peaks at about 440 nm (blue light) and 680 nm (red light), which matched the absorption spectrum of the resting spore chloroplast, as well as photosynthetic action spectra reported for other diatoms. DCMU, an inhibitor of photosynthetic electron flow near photosystem II, completely blocked photogermination. These results suggest that the photosynthetic system is involved in the photoreception process of light-induced germination. Results of pharmacological studies of the downstream signal transduction pathway suggested that Ca2+ influx is the closest downstream neighbor, followed by steps involving calmodulin, nitric oxide synthase, guanylyl cyclase, protein-tyrosine-phosphatase, protein kinase C and actin polymerization and translation

Novel photosensory two-component system (PixA-NixB-NixC) involved in the regulation of positive and negative phototaxis of cyanobacterium synechocystis sp. PCC 6803

Two wild-type substrains of a motile cyanobacterium Synechocystis sp. PCC 6803 show positive phototaxis toward a light source (PCC-P) and negative phototaxis away from light (PCC-N). In this study, we found that a novel two-component system of photoresponse is involved in the phototactic regulation. Inactivation of slr1212 (pixA), which encodes a photoreceptor histidine kinase, reverted the positive phototaxis of PCC-P to negative phototaxis, and inactivation of the downstream slr1213 (nixB) and slr1214 (nixC), which encode AraC-like transcription factor-type and PatA-type response regulators, respectively, reverted the negative phototaxis of PCC-N to positive phototaxis. Opposite effects of pixA and nixBC disruption implies an unexpected signal transduction pathway in the switching of positive and negative phototaxis. The blue/green-type cyanobacteriochrome GAF domain of PixA was expressed in Synechocystis and phycocyanobilin-producing Escherichia coli. The holoprotein covalently bound a chromophore phycoviolobilin and showed reversible photoconversion between the violet-(Pv, λ peak = 396 nm) and green-absorbing (Pg, λ peak = 533 nm) forms, although the protein from E. coli partially bound a precursor phycocyanobilin. These results were discussed with regard to an idea that PixA serves as a violet light receptor for switching of positive and negative phototaxis by transcriptional and functional regulation

Action spectrum for photoperiodic control of thyroid-stimulating hormone in Japanese quail (Coturnix japonica).

At higher latitudes, vertebrates exhibit a seasonal cycle of reproduction in response to changes in day-length, referred to as photoperiodism. Extended day-length induces thyroid-stimulating hormone in the pars tuberalis of the pituitary gland. This hormone triggers the local activation of thyroid hormone in the mediobasal hypothalamus and eventually induces gonadal development. In avian species, light information associated with day-length is detected through photoreceptors located in deep-brain regions. Within these regions, the expressions of multiple photoreceptive molecules, opsins, have been observed. However, even though the Japanese quail is an excellent model for photoperiodism because of its robust and significant seasonal responses in reproduction, a comprehensive understanding of photoreceptors in the quail brain remains undeveloped. In this study, we initially analyzed an action spectrum using photoperiodically induced expression of the beta subunit genes of thyroid-stimulating hormone in quail. Among seven wavelengths examined, we detected maximum sensitivity of the action spectrum at 500 nm. The low value for goodness of fit in the alignment with a template of retinal1-based photopigment, assuming a spectrum associated with a single opsin, proposed the possible involvement of multiple opsins rather than a single opsin. Analysis of gene expression in the septal region and hypothalamus, regions hypothesized to be photosensitive in quail, revealed mRNA expression of a mammal-like melanopsin in the infundibular nucleus within the mediobasal hypothalamus. However, no significant diurnal changes were observed for genes in the infundibular nucleus. Xenopus-like melanopsin, a further isoform of melanopsin in birds, was detected in neither the septal region nor the infundibular nucleus. These results suggest that the mammal-like melanopsin expressed in the infundibular nucleus within the mediobasal hypothalamus could be candidate deep-brain photoreceptive molecule in Japanese quail. Investigation of the functional involvement of mammal-like melanopsin-expressing cells in photoperiodism will be required for further conclusions