Dendrimer molecules with record large two-photon absorption cross section.

We report what is to our knowledge a record high value for an intrinsic two-photon absorption (TPA) cross section, sigma(2) = 11 x 10(-47)> cm>(4)> s photon(-1) molecule(-1), measured with femtosecond pulses in a new dendrimer molecule comprising 29 repeat units of 4, 4(?)-bis(diphenylamino)stilbene chromophore. We measure the dependence of TPA on excitation wavelength in three consecutive generations of the dendrimer and show that the maximum sigma(2) value increases faster than the total number of stilbene chromophores. This result indicates that it is possible to obtain even larger sigma(2) values in higher generations of this dendrimer family.

Identification, quantification, and relative concentrations of carotenoids and their metabolites in human milk and serum.

Thirty-four carotenoids, including 13 geometrical isomers and eight metabolites, in breast milk and serum of three lactating mothers have been separated, identified, quantified, and compared by high-performance liquid chromatography (HPLC)-photodiode array (PDA) detection-mass spectrometry (MS). Among the metabolites were two oxidation products of lycopene and four of lutein/ zeaxanthin. In addition, two metabolites of lutein, formed as a result of dehydration of this dihydroxycarotenoid under acidic conditions similar to those of the stomach, have also been identified in plasma and breast milk. The oxidative metabolites of lycopene with a novel five-membered-ring end group have been identified as epimeric 2,6-cyclolycopene-1,5-diols by comparison of their HPLC-UV/visible-MS profiles with those of fully characterized (1H- and 13C-NMR spectroscopy) synthetic compounds. The HPLC procedures employed also detected vitamin A, two forms of vitamin E (gamma- and alpha-tocopherol), and two non-carotenoid food components, i.e., piperine and caffeine, in serum and breast milk.

Transactivation of methylated HIV-LTR by a frog virus 3 protein.

DNA methylation has been implicated in the suppression of transcription of a large spectrum of eukaryotic genes. Frog virus 3 (FV3) contains genomic DNA that is the most extensively methylated of all known animal viruses. However, FV3 gene expression is tightly regulated in a sequential fashion in infected cells. Therefore, FV3 must have evolved a mechanism(s) to overcome the inhibitory effects of DNA methylation. FV3 has been shown to induce expression of methylated foreign genes in transient transfections. This study was designed to establish if this FV3-induced expression of methylated genes could be demonstrated in stable cell lines which contain integrated foreign genes that are silenced by DNA methylation. Stably transfected simian Vero and human T-cells containing a single copy of the methylated and transcriptionally suppressed HIV-LTR CAT construct were either infected with FV3 or fused with FV3-infected fat head minnow cells. The results from these experiments lead us to conclude that FV3 infection does promote expression of a foreign, stably integrated gene (HIV-LTR), which was previously silenced by DNA methylation. We also observed that stably transformed human T-cells incubated at 30 degrees, unlike at 37 degrees, expressed minute but detectable HIV-LTR-directed CAT activity. Significance of this finding in HIV pathogenesis remains elusive.

Interrelations of secondary structure stability and DNA-binding affinity in the bacteriophage SPO1-encoded type II DNA-binding protein TF1.

TF1, a homodimeric DNA-binding and -bending protein with a preference for hydroxymethyluracil-containing DNA is the Bacillus subtilis-encoded homolog of the bacterial HU proteins and of the E. coli integration host factor. A temperature-sensitive mutation at amino acid 25 of TF1 (L25-->A) and two intragenic second site revertants at amino acids 15 (E15-->G) and 32 (L32-->I) were previously identified and their effects on virus development were examined. The DNA-binding properties of these proteins and the thermal stability of their secondary structures have now been analyzed. Amino acids 15 and 32 are far removed from the putative DNA-binding domains of TF1 but changes there exert striking effects on DNA affinity that correlate with effects on structure. The double mutant protein TF1-G15I32 binds to a preferred site in hydroxymethyluracil-containing DNA 40 times more tightly, denatures at higher temperature (delta tm = 21 degrees C), and also exchanges subunits much more slowly than does the wild-type protein. The L25-->A mutation makes TF1 secondary structure and DNA-binding highly salt concentration-dependent. The E15-->G mutation partly suppresses this effect: secondary structure of TF1-A25G15 is restored at 21 degrees C by 1 M NaCl or, at low NaCl concentration, by binding to DNA.

Characteristics and significance of DNA binding activity of plasmid stabilization protein ParD from the broad host-range plasmid RK2.

A region of the plasmid RK2 has been shown to stabilize plasmid replicons in a broad host-range manner. This region encodes two divergently transcribed operons: parCBA and parDE. The parCBA operon specifies a multimer resolution system, while the parDE operon alone is capable of stabilizing an RK2-derived minireplicon under defined growth conditions in several different Gram-negative bacteria. The observed autoregulation of the parDE operon is most likely the result of ParD protein binding within the PparDE region. The characteristics of ParD binding to this region and the role of such binding in plasmid stabilization were examined with purified ParD protein. The results indicate that the binding of a single dimer of ParD protein to the promoter region most likely blocks interaction of RNA polymerase holoenzyme with the promoter. DNase I protection experiments indicate that ParD binds to a discrete sequence of 48 base pairs in length. While the binding of ParD to PparDE is essential for proper regulation of expression of the ParD and ParE proteins in vivo, the analyses of binding properties of mutant ParD proteins suggest that binding to this region does not play a direct role in plasmid stabilization.

In situ fluorescence cell mass measurements of Saccharomyces cerevisiae using cellular tryptophan.

This work describes a new spectroscopic optical fiber/rod technique for in situ real time measurement of cell mass and product concentrations in bioreactors using intrinsic fluorescence. The variable excitation/emission wavelength capability of this sensor allows for species-selective measurement during fermentations. Cell mass (tryptophan) and product concentrations (pyridoxine) have been measured during fermentations of Saccharomyces cerevisiae. The effects of varying substrate concentration and oxygen concentration on the observed cell mass signals are eliminated by direct measurement of cell mass, as opposed to indirect measurement schemes such as those using NADH fluorescence. The sensor is robust and able to undergo many cycles of in situ steam sterilization without degradation, and its fluorescence signal is linear with concentration for all species studied in this work. Tryptophan fluorescence from yeast is shown to be a better measure of cell mass than NADH fluorescence.

Isolation and characterization of fly ash from rat lung tissue.

Inhaled fly ash may be leached by lung fluids, making potentially toxic trace elements in the fly ash bioavailable. We studied the composition and morphology of fly ash particles recovered from lungs of rats exposed to fly ash from a power plant burning pulverized eastern coal. Animals were sacrificed 1, 3, 6, and 12 mo after the commencement of the 4-wk exposures. Particles isolated from lungs of exposed animals, control fly ash samples, and samples recovered from control lungs spiked with fly ash were characterized by computer-controlled scanning electron microscopy (CCSEM) and thin window energy dispersive X-ray spectroscopy (EDS). EDS spectra of fly ash and ashed lung residues were distinct. Thus, fly ash particles could be distinguished from ashed lung residues. A majority of the fly ash particles recovered from lungs of exposed animals had similar morphology and composition to the exposure material. However, the number of silicon-rich particles decreased with time. After 6 mo, about 1% by number of the particles had been transformed, producing numerous "needles" associated with residues of fly ash particles. Particles that looked like diatoms were observed. This demonstrated that the sample preparation procedures used did not destroy delicate structures. Fly ash particles from a spiked control lung subjected to the same separation procedures did not have these structures. The structures may be the result of leaching of particles by lung fluids, which suggests that the glassy matrix components of fly ash particles may be bioavailable.

The effects of metal ions and temperature on the interaction of cobra venom factor and human complement factor B.

An alternative pathway C3 convertase is formed by the equilibrium association of Factor B with cobra venom factor (CVF) followed by the activation step catalyzed by Factor D. However, the association of Factor B with CVF has only occasionally been demonstrated and has not been quantitatively analyzed. Here we show that in the absence of metals the two proteins have significant affinity for each other and reversibly associate in a one-to-one stoichiometry with a dissociation constant of 11.6 microM. Upon the addition of metal ions, the complex is stabilized only 2- to 30-fold in the order Ni2+(Kd = 6.6 microM) less than Mg2+(Kd = 1.1 microM) less than Mn2+(Kd = 0.4 microM). These results suggest that metal ions may be less important in stabilizing the CVF.B complex and more important in promoting the subsequent equilibrium association of CVF.B with Factor D. The stability of the CVF.B complex is variously dependent on temperature in the range studied (14-21 degrees C) depending on the metal ion that is present. The complex formation was demonstrated in the analytical ultracentrifuge at sedimentation equilibrium employing a combination of single- and multiple-independent variable nonlinear least squares analytical techniques. Two different numerical approaches gave very similar results.