Plasmonic enhanced emissions from cubic NaYF(4):Yb: Er/Tm nanophosphors.

A metal shell was used in this study to provide significant enhancement of the up-converted emission from cubic NaYF(4) nanoparticles, creating a valuable composite material for labeling in biology and other applications - use of the cubic form of the material obviates the need to undertake a high temperature transformation to the naturally more efficient hexagonal phase. The NaYF(4) matrix contained ytterbium sensitizer and an Erbium (Er) or Thulium (Tm) activator. The particle sizes of the as-synthesized nanoparticles were in the range of 20-40 nm with a gold shell thickness of 4-8 nm. The gold shell was macroscopically amorphous. The synthesis method was based on a citrate chelation. In this approach, we exploited the ability of the citrate ion to act as a reductant and stabilizer. Confining the citrate ion reductant on the nanophosphor surface rather than in the solution was critical to the gold shell formation. The plasmonic shell enhanced the up-conversion emission of Tm from visible and near-infrared regions by up to a factor of 8, in addition to imparting a visible color arising from the plasmon absorption of the gold shell. The up-conversion enhancement observed with Tm and Er were different for similar gold coverages, with local crystal field changes as a possible route to enhance up-conversion emission from high symmetry structural hosts. These novel up-converting nanophosphor particles combine the phosphor and features of a gold shell, providing a unique platform for many biological imaging and labeling applications.

Quantum Dots as Reporters in Multiplexed Immunoassays for Biomarkers of Exposure to Agrochemicals.

The application of quantum dots (QDs) as labels in immunoassay microarrays for the multiplex detection of 3-phenoxybenzoic acid (PBA) and atrazine-mercapturate (AM) has been demonstrated. PBA and AM are biomarkers of exposure to the pyrethroid insecticides and to the herbicide atrazine, respectively. Microarrays were fabricated by microcontact printing of the coating antigens in line patterns onto glass substrates. Competitive immunoassays were successfully performed using QDs (QD560 and QD620) as reporters. The multiplexed immunoassays were characterized by fluorescence microscopy and SEM. The application of QD fluorophores facilitates multiplex assays and therefore can contribute to enhanced throughput in biomonitoring.

Spatially resolved energy electron loss spectroscopy studies of iron oxide nanoparticles.

The oxidation state of iron oxide nanoparticles co-generated with soot during a combustion process was studied using electron energy-loss spectroscopy (EELS). Spatially resolved EELS spectra in the scanning transmission electron microscopy mode were collected to detect changes in the oxidation state between the cores and surfaces of the particles. Quantification of the intensity ratio of the white lines of the iron L-ionization edge was used to measure the iron oxidation state. Quantitative results obtained from Pearson's method, which can be directly compared with the literature data, indicated that the L3 /L2-intensity ratio for these particles changes from 5.5 +/- 0.3 in the particles' cores to 4.4 +/- 0.3 at their surfaces. This change can be directly related to the reduction of the iron oxidation state at the surface of the particles. Experimental results indicate that the cores of the particles are composed of gamma-Fe2O3, which seems to be reduced to FeO at their surfaces. These results were also supported by the fine structure of the oxygen K-edge and by the significant chemical shift of the iron L-edge.

Application of europium(III) chelate-dyed nanoparticle labels in a competitive atrazine fluoroimmunoassay on an ITO waveguide.

We have demonstrated the use of an optical indium tin oxide (ITO) (quartz) waveguide as a new platform for immunosensors with fluorescent europium(III) chelate nanoparticle labels (Seradyn) in a competitive atrazine immunoassay. ITO as a solid surface facilitated the successful use of particulate labels in a competitive assay format. The limit of detection in the new nanoparticle assay was similar to a conventional ELISA. The effect of particle size on bioconjugate binding kinetics was studied using three sizes of bioconjugated particle labels (107, 304, and 396nm) and a rabbit IgG/anti-IgG system in a 96-well plate. A decrease in particle size resulted in faster binding but did not increase the assay sensitivity. Flux calculations based on the particle diffusivity prove that faster binding of the small particles in this study was primarily due to diffusion kinetics and not necessarily to a higher density of antibodies on the particle surface. The results suggest that ITO could make a good platform for an optical immunosensor using fluorescent nanoparticle labels in a competitive assay format for small molecule detection. However, when used in combination with fluorescent particulate labels, a highly sensitive excitation/detection system needs to be developed to fully utilize the kinetic advantage from small particle size. Different regeneration methods tested in this study showed that repeated washings with 0.1 M glycine-HCl facilitated the reuse of the ITO waveguide.

Demonstration of droplet size and vaporization rate measurements in the near field of a two-phase jet with droplet lasing spectroscopy.

Droplet lasing spectroscopy has been applied to the measurement of droplet size and evaporation rate in a spray. A single droplet, doped with laser dye, was injected along the centerline of a liquid spray. Filters were used to block the strong elastic-scattering signal. The lasing emission from the doped droplet could be detected against the background with mass loadings of liquid in the spray as high as 20%. An analysis of the spectrum of droplet lasing was used to evaluate the droplet diameter. The evaporation rate of the droplet was obtained from consecutive lasing spectra that were obtained from the same droplet. An error analysis of the drop size and drop evaporation measurements was carried out and showed that accurate measurements of evaporation rates were feasible.

Measurement of quenching cross sections for laser-induced fluorescence of atomic arsenic.

The measurement of collisional quenching cross sections for the (4p)(2) (5s)(1) (4)P(1/2) state of atomic arsenic is reported. The arsenic (4)P(1/2) state was prepared by excitation from the (4p)(3) (4)S(3/2) ground state with 197.2-nm laser radiation. The fluorescence signal from the (4p)(2) (5s)(1) (4)P(1/2) ? (4p)(3) (2)D(1/2) transition was monitored at 249.3 nm. Quenching cross sections were obtained for hydrogen, methane, nitrogen, carbon monoxide, and ethylene.

A cellular 65-kDa protein recognizes the negative regulatory element of human papillomavirus late mRNA.

Papillomavirus late gene expression is tightly linked to the differentiation state of the host cell. Levels of late mRNAs are only in part controlled by regulation of the late promoter, other posttranscriptional mechanisms exist that reduce the amount of late mRNA in undifferentiated cells. Previously we described a negative regulatory element (NRE) located upstream of the human papillomavirus type 16 late poly(A) site. We have delineated the NRE to a 79-nt region in which a G+U-rich region was the major determinant of NRE activity. UV-crosslinking assays identified a prominent nuclear protein of 65 kDa as the only factor in close contact with the NRE, and a complex of at least five proteins, including the 65-kDa protein, was enriched on NRE-RNA. Binding of the 65-kDa protein was depleted by preincubation with poly(U) Sepharose in high salt, a property characteristic of the U2 small nuclear ribonucleoprotein auxiliary factor U2AF65 and bacterially expressed U2AF65 exhibited NRE binding. The 65-kDa protein bound to the G+U-rich NRE 3' half which shows homology to the B2P2 sequence a known U2AF65 binding site in the alpha-tropomyosin gene, and the G+U-rich element can be replaced by B2P2 in the binding assay. Treatment of cells with phorbol 12-myristate 13-acetate reduced binding of the 65-kDa protein, induced NRE binding of a cytoplasmic protein, and relieved the NRE block on reporter gene expression.

Speciation of arsenic oxides using laser desorption/ionization time-of-flight mass spectrometry.

Positive and negative ion mass spectra of arsenic trioxide (As2O3) and arsenic pentaoxide (As2O5) have been obtained by single-step laser desorption/ionization time-of-flight mass spectrometry. Pulsed UV radiation at 266 nm was used for the simultaneous desorption and ionization of the solid sample. High-mass cluster ions that are unique to the oxidation state of each oxide sample appear in the negative ion mass spectra. The As2O3 produces As3O5-, while the As2O5 yields As3O8-. The formation of unique negative cluster ions presents the capability for arsenic oxidation state speciation by laser desorption/ionization mass spectrometry. The ability of time-of-flight mass spectrometry to examine the relative amounts of each arsenic oxide present in a series of mixtures is discussed. Application of our speciation technique to a model incinerator sample is demonstrated.

Dioxinlike properties of a trichloroethylene combustion-generated aerosol.

Conventional chemical analyses of incineration by-products identify compounds of known toxicity but often fail to indicate the presence of other chemicals that may pose health risks. In a previous report, extracts from soot aerosols formed during incomplete combustion of trichloroethylene (TCE) and pyrolysis of plastics exhibited a dioxinlike response when subjected to a keratinocyte assay. To verify this dioxinlike effect, the complete extract, its polar and nonpolar fractions, some containing primarily halogenated aromatic hydrocarbons, were evaluated for toxicity using an embryo assay, for antiestrogenicity using primary liver cell cultures, and for the ability to transform the aryl hydrocarbon receptor into its DNA binding form using liver cytosol in a gel retardation assay. Each of these assays detect dioxinlike effects. Medaka (Oryzias latipes) embryos and primary liver cell cultures of rainbow trout (Oncorhynchus mykiss) were exposed to concentrations of extract ranging from 0.05 to 45 micrograms/l. Cardiotoxicity with pericardial, yolk sac, and adjacent peritoneal edema occurred after exposure of embryos to concentrations of 7 micrograms/l or greater. These same exposure levels were associated with abnormal embryo development and, at the higher concentrations, death. Some of the fractions were toxic but none was as toxic as the whole extract. In liver cells, total cellular protein and cellular lactate dehydrogenase activity were not altered by in vitro exposure to whole extract (0.05-25 micrograms/l). However, induction of cytochrome P4501A1 protein and ethoxyresorufin O-deethylase activity occurred. In the presence of whole extract, estradiol-dependent vitellogenin synthesis was reduced. Of the fractions, only fraction 1 (nonpolar) showed a similar trend, although vitellogenin synthesis inhibition was not significant. The soot extract and fractions bound to the Ah receptor and showed a significantly positive result in the gel retardation/DNA binding test. Chemical analyses using GC-MS with detection limits for 2,3,7,8-tetrachlorodibenzo-p-dioxin and dibenzofuran in the picomole range did not show presence of these compounds. Our results indicate that other chemicals associated with TCE combustion and not originally targeted for analysis may also pose health risks through dioxinlike mechanisms.

A negative regulatory element in the human papillomavirus type 16 genome acts at the level of late mRNA stability.

A negative regulatory element present in the human papillomavirus type 16 genome has been characterized. Deletion analysis has localized the 5' end of the element to the late region of the genome at the extreme 3' end of the coding region of the L1 open reading frame, around the L1 stop codon, with the element extending into the L1 3' untranslated region. For the cell lines used, the element's function was independent of cell type, tissue, or species of origin, unlike papillomavirus infection, which is very dependent on such factors. By using an mRNA decay assay, we have determined that polyadenylated RNA containing the element is much less stable than polyadenylated RNA lacking the element. This indicates that the element acts as an mRNA instability element. The significance of A-rich, GU-rich, and AUG-rich sequences for the functioning of this human papillomavirus type 16 instability element is discussed.

Analysis of human papillomavirus type 16 late mRNA 3' processing signals in vitro and in vivo.

In the human papillomavirus type 16 genome, three late mRNA putative 3' processing signals, designated LP1, LP2, and LP3, are located downstream of the late coding region. Our results show, both in vitro and in vivo, that in HeLa cells, the LP2 signal functions. Thus, the restriction in human papillomavirus type 16 late-gene expression observed in HeLa cells and other nondifferentiated epithelial cells is not achieved by regulation of late mRNA poly(A) site usage. Interestingly, alteration of three nucleotides in the GU-rich downstream sequence element converts the nonfunctional LP1 to an efficient 3' processing site, suggesting that LP1 may function in cell types other than HeLa, such as differentiated keratinocytes. Our transfection studies have identified a negative regulatory element located immediately upstream of the late mRNA 3' processing signals; this element was not associated with any alteration in 3' processing and may act as an mRNA instability element.

The Harvey ras 1 gene is activated in papillomavirus-associated carcinomas of the upper alimentary canal in cattle.

The possible activation of ras sequences in papillomavirus-associated carcinomas of the upper alimentary canal of cattle was investigated by restriction enzyme and hybridization analysis, and by DNA-mediated transformation of NIH3T3 cells. In three cancers, a squamous cell carcinoma of the palate, a squamous cell carcinoma of the rumen, and a transitional cell carcinoma of the urinary bladder, repetitive DNA sequences present in the Ha-ras 1 locus showed anomalous restriction patterns, indicating rearrangements and, in the case of the palate cancer, amplification. Genomic DNA from several cancers was capable of inducing focus formation in the NIH3T3 transformation test. DNA from primary, secondary and tertiary transformants was analysed by hybridization to bovine ras probes and by nucleotide sequencing of polymerase chain reaction products. Bovine Ha-ras 1 sequences were found in all transformants, but no nucleotide differences were detected in exon 1 or exon 2 between normal, cancer and transformed cells. It is concluded that the Ha-ras 1 gene is activated in alimentary canal carcinomas, although the activating mutation has not yet been mapped. The possible relationship between papillomavirus infection and activation of the ras gene is considered.

Human papillomavirus type 16 DNA from a vulvar carcinoma in situ is present as head-to-tail dimeric episomes with a deletion in the non-coding region.

A number of genital cancer biopsy samples were screened for the presence of human papillomavirus type 16 (HPV-16) DNA sequences. One of these samples (a vulvar carcinoma in situ) was found to contain more than 100 copies of HPV-16 DNA sequences per cell. Using this tumour DNA, a genomic library was constructed in bacteriophage lambda and the library was screened for recombinant phage containing HPV-16 sequences. Five recombinant phage clones were isolated and their DNA was analysed by restriction endonuclease digestion and blot hybridization. All five recombinants contained two copies of the HPV-16 genome present in a head-to-tail arrangement. The data are consistent with the presence of HPV-16 sequences in the tumour DNA arranged as genomic dimers in a circular episomal configuration. The HPV-16 genomes contained a deletion within the non-coding region, a region which includes the viral origin of DNA replication and transcriptional control sequences. Possible consequences of this deletion for viral replication and transcription are discussed.

The presence of bovine papillomavirus type 4 DNA is not required for the progression to, or the maintenance of, the malignant state in cancers of the alimentary canal in cattle.

In the Western Highlands of Scotland there is a very high incidence of alimentary cancers in cattle. The carcinomas of the upper alimentary canal are found in association with virus-induced benign papillomas, and transformation of papillomas to carcinomas has been observed. Strong circumstantial evidence suggests that the progression to malignancy is due to the interplay between the virus, bovine papillomavirus type 4 (BPV-4), and carcinogen(s) present in bracken fern, which infests the marginal upland grazing grounds. The carcinomas are often accompanied by adenomas and adenocarcinomas of the lower bowels. To elucidate the role of the virus in the transformation process, we have analysed several malignancies of the alimentary canal, and have detected the viral genome in only one case of transforming papilloma of the oesophagus and one case of carcinoma of the tongue. We conclude that, although required for the induction of papillomas, the presence of the BPV-4 DNA is not necessary for the progression to, or the maintenance of, the transformed state.

The phosphorylation of ribosomal protein S6 in hamster fibroblasts infected with pseudorabies virus inactivated by ultraviolet radiation.

Infection of baby hamster fibroblasts with pseudorabies virus at high multiplicities resulted in a substantial increase in the phosphorylation of ribosomal protein S6. However, the phosphorylation was still observed with virus that had been completely inactivated by u.v. irradiation. We therefore conclude that expression of the viral genome is not required for the virus to elicit this effect.

Heat shock causes diverse changes in the phosphorylation of the ribosomal proteins of mammalian cells.

When HeLa cells or BHK cells were subjected to heat shock at 42 degrees C (for 2 h) or 45 degrees C (for 10 min) there was extensive dephosphorylation of ribosomal protein S6. Concomitantly ribosomal protein L14, which is not significantly phosphorylated in normal cells, became phosphorylated, as did a non-structural protein of Mr = 27000, associated with the ribosomes. The latter effects were not prevented by cycloheximide or actinomycin D. When cells shocked at 45 degrees C for 10 min were returned to 37 degrees C for 2 h there was rephosphorylation of ribosomal protein S6 and dephosphorylation of the 27 kDa protein, but not of ribosomal protein L14.

Phosphorylation of ribosomal proteins in hamster fibroblasts infected with pseudorabies virus or herpes simplex virus.

In BHK cells infected with pseudorabies virus, there was a substantial increase in the phosphorylation of ribosomal protein S6. This increase occurred between 2 and 4 h after infection and persisted at least until 9 h. We estimated that in mock-infected cells S6 contained, on an average, one phosphate group per protein chain, whereas in infected cells this rose to between four and five phosphate groups per protein chain. A second ribosomal protein, either S16 or S18, was also phosphorylated after infection. No increase in cyclic AMP was found at the time of phosphorylation. We also found an increased phosphorylation of S6 in herpes simplex virus-infected BHK cells.

Increased phosphorylation of ribosomal protein S6 in hamster fibroblasts transformed by polyoma virus and simian virus 40.

The extent of phosphorylation of ribosomal protein S6 was compared in normal hamster fibroblasts and in fibroblasts transformed by polyoma virus or simian virus 40. In both strains of transformed cells the protein was more highly phosphorylated than in the normal cells.

The phosphorylation of protein S6 in the newly-synthesized cytoplasmic ribosomes of hamster fibroblasts.

Ribosomes were isolated from baby hamster kidney fibroblasts, either 20 min or 2 days after labelling with radioactive amino acids, and their proteins subjected to two-dimensional polyacrylamide gel electrophoresis. No significant differences were observed between the amounts of radioactivity associated with the position of the phosphorylated derivatives of protein S6. This suggests that the phosphorylation is unlikely to be important in ribosomal biogenesis or extranuclear transport.