Mori for their technical advice and assistance. Care of experimental animals was within institutional guidelines. Supported by grants from the Ministry of Education, Science, Sports and Culture of Japan, and Circulation Biosystems at the University of Tsukuba. E.T. and J.1. are research fellows of the Japan Society for the Promotion of Science.

Histone H2AX is rapidly phosphorylated in the chromatin micro-environment surrounding a DNA double-strand break (DSB). Although H2AX deficiency is not detrimental to life, H2AX is required for the accumulation of numerous essential proteins into irradiation induced foci (IRIF). However, the relationship between IRIF formation, H2AX phosphorylation (gamma-H2AX) and the detection of DNA damage is unclear. Here, we show that the migration of repair and signalling proteins to DSBs is not abrogated in H2AX(-/-) cells, or in H2AX-deficient cells that have been reconstituted with H2AX mutants that eliminate phosphorylation. Despite their initial recruitment to DSBs, numerous factors, including Nbs1, 53BP1 and Brca1, subsequently fail to form IRIF. We propose that gamma-H2AX does not constitute the primary signal required for the redistribution of repair complexes to damaged chromatin, but may function to concentrate proteins in the vicinity of DNA lesions. The differential requirements for factor recruitment to DSBs and sequestration into IRIF may explain why essential regulatory pathways controlling the ability of cells to respond to DNA damage are not abolished in the absence of H2AX.

tion and the resulting contaminating , debris are avoided. In addition, only Laser Capture Microdissection: sion the targeted of transfer cells that are affected, can approach with a 1 preci-pm. Molecular Analysis of Tissuen u s , fullv accessible the remaining for further tissue capture, on the allowing slide is coA~arative molecular analisis of adiac& cells: The exact morphology bf the pr&ured Robert F. Bonner, Michael Emmert-Buck, Kristina Cole, Thomas cells is retained andheldonthe transferfilm, Pohida, Rodrigo Chuaqui, Seth Goldstein, Lance A. Liotta so transfer images constitute a diagnostic record of the cells undergoing molecular analysis. In contrast, with manual microdissection, cells may be pulverized or lost (5).A s the list of expressed human genes ex-tion of the cells of interest. A second cat-With LCM, the procurement ofspecific cells pan&, a major scientific and medical chal-egory involves a tool under manual guidance from a complex tissue section is reduced to a lenge is to underused to mechanically separate cells of inter-routine method amenable to widespread re-TE 1 stand the molecular est from the histologic section. The tool can search and clinical diagnostic use.events that drive be a pipette, pointed probe, fine needle, or The thermoplastic polymer film contains normal tissue morphogenesis and the pro-blade (3) either hand-held or connected to a special infrared (IR) absorbing dyes to allow gression ofpathologic lesions in actual tissue. micromanipulator arm (5). Boehm et al. (6) use with near-IR gallium arsenide laser di-With the advent of polymerase chain reac-placed the tissue section on an ultrathin film odes, which are easily and economically intion (PCR) and the development of high-and then used an ultraviolet laser to cut out tegrated into normal microscopes. The polythroughput, automated microhybridization the tissue of interest. mer and its substrate do not absorb the visarrays and mutation screening methods,

A theory is developed which relates quasi-elastic light scattering measurements to blood flow in tissue micro-vasculature. We assume that the tissue matrix surrounding the blood cells is a strong diffuser of light and that moving erythrocytes, therefore, are illuminated by a spatially distributed source. Because the surrounding tissue is considered to be stationary, Doppler shifts in the frequency of the scattered light arise only from photon interactions with the moving blood cells. The theory implies that the time decay of the photon autocorrelation function scales proportionally with cell size and inversely with mean translational speed. Analysis of multiple interactions of photons with moving cells indicates the manner in which spectral measurements additionally are sensitive to changes in blood volume. Predictions are verified by measurements of particle flow in model tissues.

Measurement of gene-expression profiles using microarray technology is becoming increasingly popular among the biomedical research community. Although there has been great progress in this field, investigators are still confronted with a difficult question after completing their experiments: how to validate the large data sets that are generated? This review summarizes current approaches to verifying global expression results, discusses the caveats that must be considered, and describes some methods that are being developed to address outstanding problems.