Telomeres are essential nucleoprotein structure at the ends of all eukaryotic chromosomes. Our previous work demonstrated that mammalian telomeres were shown to end in a large t-loop structure in vitro and the formation of t-loops was dependent on the presence of TRF2. In this work, the telomere DNA and its complex of TRF2 in HeLa cells has been direct observed in the nanometer resolution regime by atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM). AFM images showed that the looped structures exited in cell extract containing TRF2, but it disappeared in the protein-deleted samples. When cells were pretreated by UV light plus psoralen, the looped structure could be observed in the protein-deleted samples. SNOM images further demonstrated TRF2 and p53 proteins in cell was bound at the loop junction. Above results suggest that the telomere t-loop structure by TRF2 play a important role in cell-senescence, and might signals p53 protein directly through association with the t-loop junction in cells.
DNA from HeLa cells was cleaved with Rasl and Hinf, and then purified by Bio-Gel P-2 column. Results showed that upon near-UV-irradiation, psoralen could be targeted to the repetitive sequences of telomeres. Large duplex loops with a tail were observed directly by AFM. The loop-tail junction position was assumed to be a tri- or tetra- strand DNA structure according to its apparent height, corresponding structure model was proposed. The psoralen cross-linking products increased with increase of irradiation time. The expression of p53 oncogene was significantly increased.
A psoralen-oligonucleotide conjugate was designed to photoinduce a cross-link at a specific sequence of c-fos oncogene. Psoralen was attached to its C-3 position of a 20-base mer oligonucleotide, which binds to a synthetic 49 bp duplex containing the c-fos gene polypurine site, where it forms a triple stranded DNA. Upon near-UV-irradiation, the two strand of DNA are crosslinked at the TpA step present at the triple-duplex junction. Results show that the yield of the photoinduce cross- linking reaction is quite high. We treated HeLa cells with above 2-mer oligonucleotide conjugated to psoralen. The expression of c-fos oncogene was significant reduced, no significant effect on the level of c-myc mRNA. These data indicate that such psoralen- oligonucleotide conjugates could be used to selectively control gene expression or to induce sequence-specific damages.
The application of image fusion in biomedical image analysis has led to a new concept about the nature of disease and to new diagnostic capabilities. However, the algorithm research of fusion is still an open research topic because the algorithm is often changed with different original images, different detectors and different research objects. This paper focuses on comparison of four image fusion algorithms based on wavelet transform to select the suitable methods for biomedical image fusion. The algorithms include: (1) weighted algorithm, (2) maximum selection algorithm, (3) stressing one image and (4) logic OR algorithm. For the sake of selecting the suitable image fusion methods for biomedical images, we propose six quantitative performance measure criterion linked with the characters of the biomedical image: standard deviation ((sigma) ), peak signal-to-noise ratio (PSNR), mean deviation ((Delta) (mu) ), the difference in entropy ((Delta) H), coefficient of correlation between the fusion image and ideal image (Correlation) and the difference in contrast between the fusion image and ideal image ((Delta) Contrast). Using the four algorithms to process biomedical images, such as fluorescence image and the corresponding transmission image, the visual investigation and the six quantitative performance measure criterion indicate that the weighted algorithm is the most suitable method for biomedical images among the four algorithms.
PDT-induced HeLa cells apoptosis was evidenced by morphological changes, DNA fragmentation and decrease of mitochondria dehydrogenase activity. Apoptosis was associated with the formation of IP<SUB>3</SUB> of photosensitized cells and rapid release of Ca<SUP>2+</SUP> from intracellular pools. Vitamin E, which was added before irradiation, could significantly protect cells against cell death, indicating that reactive oxygen species (ROS) play an important role in apoptotic cell death.
Exposure of thymine and DNA to high-intensity 532 nm pulsed radiation from a Nd:YAG laser resulted in the cyclobutylpyrimidine dimers, which were measured by the method of high performance liquid chromatography. The in vitro transcription by RNA polymerase was markedly inhibited and could not be stimulated by spermine when the native double-strand DNA was replaced by irradiated DNA. It was shown that DNA damage was caused by 532 nm laser radiation and that the high-intensity visible radiation can initiate photochemistry in UV-absorbing biological molecules by two photon absorption. It is suggested that the use of very high-intensity laser radiation in medicine introduces the possibility that biomacromolecules may be damaged in cells as a result of two photon absorption.
Hypocrellin A (HC-A) isolated from Hypocrella Membrane Sacc, a derivative of 3,10-dihydroxy-4, 9-peryleneguinone, is a new effective photosensitizer. Treatment of the CESS cell with 20 (mu) g per ml of HC-A plus laser (532 nm) radiation led to the formation of DNA single strand break. The number of single strand breaks for cell membrane DNA was approximately 2.5 times that of nuclear DNA. Treatment of the CESS cell with HC-A plus laser radiation reduced the cell DNA binding capacity by 59%. It is shown that cell membrane DNA is also a target for HC-A photosensitization.