AU-rich elements (AREs) in the 3' untranslated region (UTR) of numerous mammalian transcripts function as instability elements that promote rapid mRNA degradation. Tristetraprolin (TTP) is an ARE-binding protein that promotes rapid mRNA decay through mechanisms that are poorly understood. A 31 nucleotide ARE sequences from the TNF-alpha 3' UTR promoted TTP-dependent mRNA decay when it was inserted into the 3' UTR of a beta-globin reporter transcript, indicating that this short sequence was sufficient for TTP function. We used a gel shift assay to identify a TTP-containing complex in cytoplasmic extracts from TTP-transfected HeLa cells that bound specifically to short ARE sequences. This TTP-containing complex also contained the 5'-3' exonuclease Xrn1 and the exosome component PM-scl75 because it was super-shifted with anti-Xrn1 or anti-PMscl75 antibodies. RNA affinity purification verified that these proteins associated specifically with ARE sequences in a TTP-dependent manner. Using a competition binding assay, we found that the TTP-containing complex bound with high affinity to short ARE sequences from GM-CSF, IL-3, TNF-alpha, IL-2, and c-fos, but did not bind to a U-rich sequence from c-myc, a 22 nucleotide poly U sequence or a mutated GM-CSF control sequence. High affinity binding by the TTP-containing complex correlated with TTP-dependent deadenylation and decay of capped, polyadenylated transcripts in a cell-free mRNA decay assay, suggesting that the TTP-containing complex was functional. These data support a model whereby TTP functions to enhance mRNA decay by recruiting components of the cellular mRNA decay machinery to the transcript.

Since organelle preparations often contain more than one organelle type (e.g., acidic organelles and mitochondria), techniques that measure the properties of a given organelle type while avoiding biases caused by ancillary subcellular compartments are highly desirable. We report here the use of capillary electrophoresis (CE) with laser-induced fluorescence (LIF) dual-channel detection to identify acidic organelles containing doxorubicin (DOX) in crude subcellular fractions from CCRF-CEM and CEM/C2 cell lines. As confirmed by confocal microscopy, acidic organelles are identified by their accumulation of fluorescently labeled nanospheres. Using CE-LIF analysis, individually detected organelles are classified into three kinds: acidic organelles containing only nanospheres, acidic organelles containing nanospheres and DOX, and other organelles containing DOX (e.g., mitochondria) with no detectable nanospheres. Electrophoretic mobility, DOX fluorescence intensity, and nanosphere fluorescence intensity distributions of individual acidic organelles and other organelles containing DOX are determined in the same CE-LIF run. The acidic organelle mobilities range from (-0.7 to -2.0) x 10(-4) cm(2) V(-1) s(-1) while those of the other organelles spread from (-0.6 to -3.5) x 10(-4) cm(2) V(-1) s(-1). In addition, by calibrating the detector response, DOX content in individual acidic organelles and other organelles can be estimated. The average amounts of DOX per acidic organelle in CEM/C2 and CCRF-CEM cells are 11.1 +/- 0.5 and 10.6 +/- 0.4 zmol, respectively. This first report of an analysis of the accumulation of DOX in individual acidic organelles presents a procedure for analyzing the accumulation of fluorescent compounds in acidic organelles that could be useful for investigating acidic organelle maturation and the role of these organelles in drug resistance.

Porous alumina lters were coated by vacuum deposition with silver metal and used as substrates for surface-enh anced Raman scattering (SERS). These were studied by UV-Vis absorption spectro scopy and scanning electron microscopy. The substrates exhibit reproducible Raman enhancement for different m olecules tested in this study. The mass thickness of the silver was optimized for maximal SERS signal using bipyridine as a model compound. Plasma treatment of the substrates prior to SERS measurements was identi ed as a crucial factor for a low background and high signal-to-noise Raman spectra. The effect of different plasma treatm ent conditions on the SERS signal as well as on UV-Vis absorption spectra of the substrates was investigated. After the silver deposition, the porous alumina retained its ltering abilities and could be used for preconcentrating dilute analytes on the substrate surface for SERS measurements. Corresponding tests were performed using benzotriazole and bipyridine solutions. These substrates demonstrate a potential for large-scale applications in different analytical measurem ents.

Results are presented of nonphotochemical-hole-burning experiments on the mitochondrial specific dye rhodamine 800 incubated with two human ovarian surface epithelial cell lines: OSE(tsT)-14 normal cells and OV167 carcinoma cells. This dye is selective for the plasma and inner membranes of the mitochondria, as shown by confocal microscopy images. Dispersive hole-growth kinetics of zero-phonon holes are analyzed with theoretical fits, indicating that subcellular structural heterogeneity of the carcinoma cell line is lower relative to the analogous normal cell line. Broadening of holes in the presence of an applied electric field (Stark effect) was used to determine the permanent dipole moment change for the S(0)-->S(1) transition in the two cell lines. For the carcinoma cell line, the permanent dipole moment change value is a factor of 1.5 higher than for the normal cell line. It is speculated that this difference may be related to differences in mitochondrial membrane potentials in the two cell lines.