These cell lines are the first available primary epithelial and stromal cells derived from an African-American patient with organ-confined prostate cancer and in conjunction with other established cell lines, could provide an in vitro model system to investigate early transforming events in prostate cancer.

Exposure of a single crystal of the nitrite adduct of ferric myoglobin (Mb) at 100 K to highintensity synchrotron X-ray radiation resulted in changes in the UV-vis spectrum that can be attributed to reduction of the ferric compound to the ferrous derivative. We employed correlated single-crystal spectroscopy with crystallography to further characterize this photoproduct. The 1.55 Å resolution crystal structure of the photoproduct reveals retention of the O-binding mode of nitrite to the iron center. The data are consistent with the cryogenic generation and trapping, at 100 K, of a ferrous d 6 Mb II (ONO)* complex by photoreduction of the ferric precursor crystals using high-intensity X-ray radiation.Some mammalian proteins such as the heme-containing myoglobin (Mb) can reduce the nitrite anion (NO 2 − ; pK a 3.2) under hypoxic conditions to nitric oxide (NO; eq 1) (1,2) in a process somewhat reminiscent of that employed by denitrifying nitrite reductase (NiR) enzymes.(1)Although this subject area is under active investigation and debate, Gladwin and coworkers have demonstrated that nitrite protects against myocardial infarction in Mb +/+ mice, but does not in Mb −/− knockout mice, thus indeed implicating Mb as an in vivo NiR (1).Clearly, the ability of heme to facilitate nitrite reduction (eq 1) necessitates its presence in the ferrous form that can supply the electron needed for this process. We reported the X-ray crystal structure of the related stable ferric d 5 Mb-nitrite compound, and showed that the † This work was supported by the Oklahoma Center for the Advancement of Science and Technology (HR9-081, to GBR-A) and by SUPPORTING INFORMATION AVAILABLE X-ray data collection, structure solution and refinement; Movie in avi format showing the single-crystal spectra before and after highintensity X-ray exposure (for the process described in Fig. 4) as a function of crystal rotation angle. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 July 27. (4), and this nitro mode was observed in the X-ray crystal structures of the nitrite adducts of cyt cd 1 NiR (P. pantotrophus (5)), sulfite reductase hemoprotein (E. coli (6)), and cyt c NiR (W. succinogenes (7) and T. nitratireducens (8)). In contrast, the nitrito mode was observed in the nitrite adduct of ferric human hemoglobin (9).We were unable, however, and despite several varied attempts, to generate the physiologically important ferrous d 6 wild-type Mb-nitrite derivative to determine the nitrite binding mode (3). We have been intrigued by the use of synchrotron X-ray radiation for the generation of electrons in protein crystals (10). We have thus taken advantage of correlated microspectrophotometry and synchrotron X-ray photoreduction techniques to generate and characterize a ferrous d 6 Mb-nitrite compound at 100 K.Single crystals of ferric horse heart Mb III (ONO) at pH 7.4 were generated as previously described (3). The single...

STRATEGY is a program for determination of the optimal starting spindle angle and scan range for X-ray diffracton data collection. It is optimized for crystals of macromolecular compounds using a one-circle diffractometer with a twodimensional detector. In its current implementation, the program reads the starting crystal orientation and cell parameters from a DENZO intensities '.x' file and simulates all the reflections that can occur during a 360 ° rotation of the crystal. It determines whether the reflections can be recorded on the detector and sorts them on spindle angle. Then it gives diagrams representing the needed sweep as a function of the starting spindle angle for different degrees of completeness of the data set wanted, and produces redundancy tables for the shortest data collection possible for each desired completeness. Use of the program assists in careful planning of data collection: it will assure a complete data set, and certainly will allow data collection in the shortest possible time, sometimes reducing the data-collection time by a factor of two or more, depending on the lattice symmetry and the data-collection mode.

We demonstrate a general strategy to determine structures from showers of microcrystals. It uses acoustic droplet ejection (ADE) to transfer 2.5 nanoliter droplets from the surface of microcrystal slurries, through the air, and onto mounting micromesh pins. Individual microcrystals are located by raster-scanning a several micron X-ray beam across the cryocooled micromeshes. X-ray diffraction datasets merged from several micron-sized crystals are used to solve 1.8 Å resolution crystal structures.

Micro-sized polyimide well-mounts for the manipulation of microcrystals and a data-assembly method for rotation data sets from many microcrystals are described.