Manipulation of cells with laser microbeam scissors and optical tweezers: a review.

The use of laser microbeams and optical tweezers in a wide field of biological applications from genomic to immunology is discussed. Microperforation is used to introduce a well-defined amount of molecules into cells for genetic engineering and optical imaging. The microwelding of two cells induced by a laser microbeam combines their genetic outfit.

Butyrate modulates antioxidant enzyme expression in malignant and non-malignant human colon tissues.

The induction of antioxidant enzymes is an important mechanism in colon cancer chemoprevention, but the response of human colon tissue to butyrate, a gut fermentation product derived from dietary fiber, remains largely unknown. Therefore, our study investigated the effect of a butyrate treatment on catalase (CAT) and superoxide dismutase (SOD2) in matched human colon tissues of different transformation stages (n = 3-15 in each group) ex vivo. By performing quantitative real-time PCR, Western blot, and spectrophotometric measurements, we found an increase in SOD2 at expression and activity level in colonic adenocarcinomas (mRNA: 1.

Poly(ADP-ribose) binding to Chk1 at stalled replication forks is required for S-phase checkpoint activation.

Damaged replication forks activate poly(ADP-ribose) polymerase 1 (PARP1), which catalyses poly(ADP-ribose) (PAR) formation; however, how PARP1 or poly(ADP-ribosyl)ation is involved in the S-phase checkpoint is unknown. Here we show that PAR, supplied by PARP1, interacts with Chk1 via a novel PAR-binding regulatory (PbR) motif in Chk1, independent of ATR and its activity. iPOND studies reveal that Chk1 associates readily with the unperturbed replication fork and that PAR is required for efficient retention of Chk1 and phosphorylated Chk1 at the fork.

Proteomic identification of PSF and p54(nrb) as TopBP1-interacting proteins.

TopBP1 is a BRCT domain-rich protein that is structurally and functionally conserved throughout eukaryotic organisms. It is required for the initiation of DNA replication and for DNA repair and damage signalling. To further dissect its biological functions, we explored TopBP1-interacting proteins by co-immunoprecipitation assays and LC-ESI-MS-analyses.

Impact of butyrate on PKM2 and HSP90β expression in human colon tissues of different transformation stages: a comparison of gene and protein data.

Due to protection of oncogenic proteins from degradation and enhancement of glycolytic phosphometabolites for synthetic processes, respectively, heat shock protein 90 (HSP90) and pyruvate kinase type M2 (PKM2) are important proteins for tumor growth. The present study was undertaken to investigate the susceptibility of both proteins and their encoding genes to the chemopreventive agent butyrate in human colon cells. Matched tissue of different transformation stages derived from 20 individual colon cancer patients was used for the experiments.

Photons bring light into DNA repair: the comet assay and laser microbeams for studying photogenotoxicity of drugs and ageing.

This contribution reviews recent applications of micromanipulation, by UV photons, in DNA repair and ageing research as well as in the evaluation of the phototoxicity of drugs. In some cases, micromanipulation is combined with the comet assay, a technique, which allows a direct view on DNA damages. It is shown that, in humans, the sensitivity of DNA to UV induced damage and its subsequent repair is surprisingly stable up to high age and that drugs which are usually non-toxic induce DNA damage when irradiated in parallel by UV irradiation.

Defensin alpha 6 (DEFA 6) overexpression threshold of over 60 fold can distinguish between adenoma and fully blown colon carcinoma in individual patients.

Background: It is known that alpha-defensin expression is enhanced in colon cancer. However, the expression of human alpha defensin 6 (DEFA 6) in earlier stages, such as adenoma, has so far not yet been studied in a patient resolved manner.

Methods: By using quantitative Real Time-PCR, the gene expression pattern of DEFA 1-3 and DEFA 6 was analyzed in tissue of different stages of carcinogenesis, derived from colorectal cancer patients.

Single molecule experiments challenge the strict wave-particle dualism of light.

Single molecule techniques improve our understanding of the photon and light. If the single photon double slit experiment is performed at the "single photon limit" of a multi-atom light source, faint light pulses with more than one photon hamper the interpretation. Single molecules, quantum dots or defect centres in crystals should be used as light source.

A direct view by immunofluorescent comet assay (IFCA) of DNA damage induced by nicking and cutting enzymes, ionizing (137)Cs radiation, UV-A laser microbeam irradiation and the radiomimetic drug bleomycin.

In DNA repair research, DNA damage is induced by different agents, depending on the technical facilities of the investigating researchers. A quantitative comparison of different investigations is therefore often difficult. By using a modified variant of the neutral comet assay, where the histone H1 is detected by immunofluorescence [immunofluorescent comet assay (IFCA)], we achieve previously unprecedented resolution in the detection of fragmented chromatin and show that trillions of ultraviolet A photons (of a few eV), billions of bleomycin (BLM) molecules and thousands of gamma quanta (of 662 keV) generate, in first order, similar damage in the chromatin of HeLa cells.

Laser microbeams and optical tweezers in ageing research.

We show how a technique developed within the framework of physics and physical chemistry-in a true interdisciplinary approach-can answer questions in life sciences that are not solvable by using other techniques. Herein, we focus on blood-pressure regulation and DNA repair in ageing studies. Laser microbeams and optical tweezers are now established tools in many fields of science, particularly in the life sciences.

Repair of sparfloxacin-induced photochemical DNA damage in vivo.

The induction and subsequent repair of photochemically induced DNA damage by sparfloxacin was assessed in different tissues of juvenile Wistar rats. The animals were treated once orally with 500 mg kg(-1) of sparfloxacin and irradiated 3 hours later with 7 J cm(-2) UVA. Induction and repair of DNA damage was studied in the skin, retina and cornea using the alkaline comet assay.

Detection of photogenotoxicity in skin and eye in rat with the photo comet assay.

Photosensitizing drugs increase the sensitivity of the skin and the eye toward normally harmless sunlight conditions and are known to enhance the induction of skin tumors or severe injuries to the eye. The photogenotoxicity of five common drugs (sparfloxacin, dacarbazine, chlorpromazine and 8-methoxypsoralen, promazine) was investigated in the skin as well as in the retina and cornea of Wistar rats. The compounds were administered once orally by gavage and the resulting DNA damage was analyzed in the newly developed in vivo photo comet assay.

Orientation of erythrocytes in optical trap revealed by confocal fluorescence microscopy.

There has been considerable current interest in the rotational behavior of red blood cells (RBCs) in optical tweezers. However, the mechanism of rotation in polarized tweezers is still not well understood and conflicts exist in the understanding of this phenomenon. Therefore, we reexamined the underlying phenomenon by use of confocal fluorescence microscopy in combination with optical tweezers.

Selected applications of laser scissors and tweezers and new applications in heart research.

This contribution bridges the gap from early European contributions via laser micromanipulation to recent work on the use of laser microbeams and optical tweezers in studies of basic aspects of heart infarction. Laser transfection, particularly of plant cells and their chloroplasts, and laser microdissection of chromosomes with subsequent generation of chromosome segment-specific DNA libraries and laser-induced cell fusion are reported. With optical tweezers, microgravity can be simulated in roots of the alga Chara.

The photo comet assay--a fast screening assay for the determination of photogenotoxicity in vitro.

Different classes of chemicals can induce a phototoxic effect by absorbing light energy within the wavelength range of sunlight. The assessment of photo-safety is therefore an obligatory part of the development of new drugs. Ten UV-vis (280-800nm)-absorbing compounds (ketoprofen, promazine, chlorpromazine, dacarbazine, acridine, lomefloxacin, 8-methoxypsoralen, chlorhexidine, titanium dioxide, octylmethoxycinnamate) were tested for their photogenotoxic potential in the alkaline comet assay in the presence and absence of UV-vis.

Endosymbiont-dependent host reproduction maintains bacterial-fungal mutualism.

Bacterial endosymbionts play essential roles for many organisms, and thus specialized mechanisms have evolved during evolution that guarantee the persistence of the symbiosis during or after host reproduction. The rice seedling blight fungus Rhizopus microsporus represents a unique example of a mutualistic life form in which a fungus harbors endobacteria (Burkholderia sp.) for the production of a phytotoxin.

Near-field scanning optical microscopy in cell biology and cytogenetics.

Light microscopy has proven to be one of the most versatile analytical tools in cell biology and cytogenetics. The growing spectrum of scientific knowledge demands a continuous improvement of the optical resolution of the instruments. In far-field light microscopy, the attainable resolution is dictated by the limit of diffraction, which, in practice, is about 250 nm for high-numerical-aperture objective lenses.

Single-molecule studies on DNA and RNA.

DNA and RNA are the most individual molecules known. Therefore, single-molecule experiments with these nucleic acids are particularly useful. This review reports on recent experiments with single DNA and RNA molecules.

Ubiquitous cancer genes: multipurpose molecules for protein micro-arrays.

Multipurpose genes in the human genome which are over-expressed in a large variety of different cancers have been identified. Forty-two of the 19,016 human genes annotated to date (0.2%) are ubiquitously over-expressed in half or more of the 36 investigated human cancers.

Assessment of DNA damage in target tumor cells after thermoablation in mice.

Purpose: To determine the effects of temperature on cell death for cells in culture and to compare these effects with the results of in vivo experiments in which heating is induced in mice with implanted human adenocarcinoma by using magnetic methods.

Materials And Methods: Experimentation was approved by the regional animal care committee. Human adenocarcinoma cells (MX-1) and human fibroblasts (HTB-125) were exposed to defined temperatures of 45 degrees -90 degrees C for 4 minutes.

Fragility of telomeres after bleomycin and cisplatin combined treatment measured in human leukocytes with the Comet-FISH technique.

Unlabelled: THE AIM of the present study was the comparative investigation of action of widely applied anticancer preparations: cisplatin (cis-DDP) and bleomycin (BLM) on total DNA and telomeres damage in human blood cells.

Methods: The "Comet-FISH technique" -- single cell gel electrophoresis ("comet assay") in combination with fluorescent in situ hybridization (FISH) was used for this purpose. This newly applied combined approach permits to detect on the same specimen the total DNA damage in individual cells and evaluate specific DNA sequences as well.

Spatial association of homologous pericentric regions in human lymphocyte nuclei during repair.

Spatial positioning of pericentric chromosome regions in human lymphocyte cell nuclei was investigated during repair after H(2)O(2)/L-histidine treatment. Fifteen to three-hundred minutes after treatment, these regions of chromosomes 1, 15, and X were labeled by fluorescence in situ hybridization. The relative locus distances (LL-distances), the relative distances to the nuclear center (LC-distances), and the locus-nuclear center-locus angles (LCL-angles) were measured in approximately 5000 nuclei after two-dimensional microscopy.