Stimulatory effect of allantoin on imidazoline I1 receptors in animal and cell line.

Allantoin is known as the agonist of imidazoline receptor, especially the I2 subtype. Effect of allantoin on imidazoline I1 receptor (I1R) relating to reduction of blood pressure and its merit in steatosis are still obscure. Also, farnesoid X receptor (FXR) plays an important role in lipid homeostasis related to I1R activation. Thus, we administered allantoin into high fat diet (HFD)-fed mice showing hypertriglyceridemia and hypercholesterolemia. Allantoin significantly improved hyperlipidemia in HFD mice after 4 weeks of administration. Pretreatment with efaroxan, at a dose sufficient to inhibit I1R activation, attenuated the action of allantoin. In addition, in cultured HepG2 cells, allantoin increased the expression of farnesoid X receptor (FXR). The allantoin-induced FXR expression was blocked by efaroxan. Similar changes were observed in the expressions of FXR-targeted genes. Otherwise, allantoin also lowered systolic blood pressure (SBP) in HFD mice that can be blocked by efaroxan. Taken together, allantoin has an ability to activate I1R for improvement of metabolic disorders.

Sugar additives for MALDI matrices improve signal allowing the smallest nucleotide change (A:T) in a DNA sequence to be resolved.

Sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) of DNA is critical for obtaining high quality mass spectra. Sample impurity, solvent content, substrate surface and environmental conditions (temperature and humidity) all affect the rate of matrix-analyte co-crystallization. As a result, laser fluence threshold for desorption/ionization varies from spot to spot. When using 3-hydroxypicolinic acid (3-HPA) as the matrix, laser fluence higher than the threshold value reduces mass resolution in time-of-flight (TOF) MS as the excess energy transferred to DNA causes metastable decay. This can be overcome by either searching for 'hot' spots or adjusting the laser fluence. However, both solutions may require a significant amount of operator manipulation and are not ideal for automatic measurements. We have added various sugars for crystallization with the matrix to minimize the transfer of excess laser energy to DNA molecules. Fructose and fucose were found to be the most effective matrix additives. Using these additives, mass resolution for DNA molecules does not show noticeable deterioration as laser energy increases. Improved sample preparation is important for the detection of single nucleotide polymorphisms (SNPs) using primer extension with a single nucleotide. During automatic data acquisition it is difficult to routinely detect heterozygous A/T mutations, which requires resolving a mass difference of 9 Da, unless a sugar is added during crystallization.

Expression immunoassay.

Expression immunoassay is a sensitive analytical method that takes advantage of coupled in vitro transcription and translation as a signal amplification technique. Essentially, the immunoassay is performed using a detection antibody that is labeled with an expressible fragment of DNA. The product of expression is a protein that can be used to generate a signal. Here we describe two distinct expression immunoassays; both are based on expression of DNA labels to produce active enzyme molecules which are subsequently detected through their enzymatic activities. The luciferase expression immunoassay uses a 2.1-kb DNA template as a reporter molecule. The DNA is attached to a biotinylated detection antibody via biotin-streptavidin linkage. After the detection antibody is immunoreacted with immobilized antigen and excess antibody is removed, the DNA label is expressed in vitro. A linear relationship exists between the bioluminescent signal, from luciferase activity, and the immobilized antigen. This expression immunoassay allows the detection of 5 x 10(4) antigen molecules. The second expression immunoassay makes use of the well-characterized alpha-complementation of beta-galactosidase by employing an alpha-peptide encoding DNA as a reporter molecule. By monitoring the resulting beta-galactosidase activity with a fluorogenic substrate it was possible to detect as little as 3 fmol of immobilized antigen. Both expression immunoassays are amenable to automation and demonstrate the potential sensitivity that can be achieved using in vitro expression as a signal amplification method.

Mass spectrometry of single-stranded restriction fragments captured by an undigested complementary sequence.

In this report, we describe a simple and accurate method to analyze restriction fragments using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The two complementary strands of restriction fragments are separated through hybridization to a capture probe, which is a single-stranded undigested fragment. Using the biotin-streptavidin linkage, the hybrid is immobilized on streptavidin-coated magnetic beads. After conditioning the captured restriction fragments, they are eluted from the probe and their molecular weights are determined. The proposed method greatly improves the quality, and reduces the complexity of the mass spectrum by analyzing only one of the complementary strands of restriction fragments.

Automated mass spectrometry: a revolutionary technology for clinical diagnostics.

For various diagnostic analyses and the studies of functional genomics, the use of an accurate and cost-effective analytic platform to analyze large numbers of samples is essential. An automated platform called MassArray (Sequenom, Inc, San Diego, CA), designed for high-throughput diagnostic analyses, has recently been validated. The platform combines miniaturized, two-dimensional chip arrays with proven high-fidelity enzymatic procedures and matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Nanoliter dispensing of samples in high-density formats of 384 or greater results in improved throughput and reduced costs. Automation prevails from the initial assay design through sample processing and data analysis, for the most part eliminating the labor component of assay development and implementation. The MassArray platform is being used in the following areas: (1) molecular diagnosis of genetic disease and infectious agents, (2) pharmacogenomics, (3) paternity and/or identity testing, and (4) agriculture (e.g., marker-assisted breeding). MALDI-TOF mass spectrometry can also be used for analyzing proteins; therefore, genotype/phenotype testing can be performed on a single platform.

Two-site expression immunoassay using a firefly luciferase-coding DNA label.

BACKGROUND: We report the first two-site, "sandwich type" expression immunoassay using as a label an expressible DNA fragment encoding firefly luciferase. METHODS: The DNA label consisted of a T7 RNA polymerase promoter, a firefly luciferase-coding sequence, and a poly(dA/dT) tail. The 3' end of the DNA label was biotinylated and complexed with streptavidin. A sandwich immunoassay for prostate-specific antigen (PSA) was developed in which the antigen was first bound to an immobilized monoclonal antibody and then reacted with a biotinylated polyclonal antibody. The streptavidin-luciferase-coding DNA complex was then bound to the immunocomplex. The DNA label was subsequently expressed in vitro by coupled transcription and translation. The generated luciferase was measured by its characteristic bioluminescent reaction. RESULTS: The bioluminescence was linearly related to the concentration of PSA in the sample. As low as 30 ng/L PSA was measured (12.5-microL sample) with a signal-to-background ratio of 2.3, and the linear range extended to 3 microg/L. The results obtained from the proposed assay agreed well to those determined by IMx immunoassay (y = 0.98x + 0.74 microg/L; r = 0.971; n = 44). CONCLUSIONS: The use of the newly developed DNA label in a two-site immunoassay was demonstrated for the first time. The assay was applied successfully to the measurement of serum PSA.

Expression immunoassay based on antibodies labeled with a deoxyribonucleic acid fragment encoding the alpha-peptide of beta-galactosidase.

An immunoassay is reported which uses, as a label, an expressible DNA fragment encoding the alpha-peptide of beta-galactosidase. This inactive peptide consists of 97 amino acid residues containing an amino-terminal portion of the enzyme. Antigen (an anti-thyrotropin immunoglobulin) immobilized in microtiter wells is allowed to react with specific antibodies which are then linked to the DNA label via biotin-streptavidin interaction. After completion of the immunoreaction, the solid phase bound DNA is subjected to a cell-free, one-step transcription/translation reaction to produce the alpha-peptide. The alpha-peptide is allowed to react (complementation reaction) with the remaining part of the beta-galactosidase (M15 protein, also inactive) to give fully active enzyme molecules. 4-Methylumbelliferyl galactoside is used as a substrate. The fluorescence is linearly related to the amount of antigen in the well. As little as 3 fmol of antigen can be detected. The RSDs (within-run) obtained for 8 and 20 fmol of antigen were 10.7 and 9.3%, respectively (n = 4). The present work illustrates the utility of expressing a non-detectable peptide capable of triggering a signal generating system.

Sandwich-type deoxyribonucleic acid hybridization assays based on enzyme amplified time-resolved fluorometry.

We report microtiter well-based sandwich-type DNA hybridization assays using enzyme amplified time-resolved fluorometry of Tb3+ chelates. The target DNA was hybridized with two adjacent and non-overlapping oligonucleotide probes, one oligonucleotide serving as the capture probe and the other as the detection probe. Two ligand-specific binding protein pairs were used alternately for capture of the hybrids to the solid phase and detection; the biotin-streptavidin and the digoxigenin-anti-digoxigenin interaction. In both cases, alkaline phosphatase was used as a reporter molecule and diflunisal phosphate as a substrate. The catalytic hydrolysis of the substrate produces diflunisal which forms ternary fluorescent complex with Tb(3+)-EDTA. Furthermore, we studied the effect of the probe labeling method and the position of the label on the sensitivity of the assays. The data suggest that capture of the hybrids through biotin-streptavidin and detection via digoxigenin-anti-digoxigenin offer 2-3 times higher sensitivity than the reverse configuration. The highest sensitivity was achieved with enzymatic labeling of capture and detection probes at the 3' end. A signal-to-background ratio of 4 was achieved for 0.2 fmol of target DNA. The RSD were better than 4%.

Hybridization assays using an expressible DNA fragment encoding firefly luciferase as a label.

We report the use of a new label, an expressible enzyme-coding DNA fragment, for nucleic acid hybridization assays. The DNA label contains a firefly luciferase coding sequence downstream from a T7 RNA polymerase promoter. The target DNA (200 bp) is denatured and hybridized simultaneously with two oligonucleotide probes. One of the probes is immobilized in microtiter wells, via the digoxigenin/anti-digoxigenin interaction, and the other probe is biotinylated. After completion of the hybridization, the hybrids are reacted with a streptavidin-luciferase DNA complex. Subsequently, the solid-phase bound DNA is expressed by coupled transcription/ translation. The synthesized luciferase catalyzes the luminescent reaction of luciferin with O2 and ATP. The luminescence is linearly related to the amount of target DNA in the range of 5-5000 amol. The CVs obtained for 20 and 100 amol of target are 6.5% and 10.8%, respectively (n = 4).

Expression immunoassay. Antigen quantitation using antibodies labeled with enzyme-coding DNA fragments.

A novel immunoassay is reported which uses an enzyme-coding DNA fragment as label (expression immunoassay). The DNA label is determined with high sensitivity by measuring the enzymatic activity produced after expression. A DNA fragment encoding the firefly luciferase is biotinylated and complexed with streptavidin. Biotinylated, specific antibodies are used for quantitation of antigen immobilized on microtiter wells. After completion of the immunoreaction, streptavidin-DNA is bound to the immunocomplex. Subsequent expression of the solid phase-bound DNA, by an one-step (coupled) cell-free transcription/translation, produces luciferase. The enzyme catalyzes the luminescent reaction of luciferin with O2 and ATP. As few as 3000 molecules of DNA label can be detected. Also, 50,000 antigen molecules can be detected, and the luminescence is a linear function of the number of antigen molecules in a range extending over 3 orders of magnitude. The high sensitivity achieved is a result of the combined amplification due to transcription/translation and the substrate turnover.

Flat revertants of temperature-insensitive transformants induced by simian virus 40 tsA mutants lose their ability to express T-antigen.

Temperature-insensitive transformants that contained simian virus 40 sequences at only one or a few sites in the rat chromosome and that were induced by a temperature-sensitive A gene mutant of simian virus 40 were used to select flat revertants (revertants that had lost the transformed phenotype). The isolation was performed at the nonpermissive temperature so as not to select against temperature-sensitive transformants. Nonetheless, all of the revertants examined had lost their ability to express the T-antigen at both temperatures, and all contained rearrangements of the integrated simian virus 40 sequences. These results are most compatible with the hypothesis that the T-antigen of simian virus 40 is required for the maintenance of the transformed state even in temperature-insensitive cell lines.

Evidence for the role of double-helical structures in the maturation of simian virus-40 messenger RNA.

Simian Virus-40 infected BSC-1 cells were pretreated with glucosamine and briefly pulsed with [3H]-uridine. The labeling can be halted instantaneously by the addition of cold uridine and glucosamine. Under these pulse-chase conditions, the inhibitory effects of the intercalating agent proflavine on the processing of prelabeled nuclear RNA precursors were examined in vivo. Proflavine inhibits the cleavage of viral nuclear RNA precursors. However, turnover of the mature viral mRNAs in the cytoplasm is not inhibited. The effect of proflavine on processing is not a secondary consequence of its inhibition of protein synthesis. The data suggest that base-paired secondary structures in the primary transcripts are important processing signals in the generation of viral mRNA molecules.

Selective degradation of newly synthesized nonmessenger simian virus 40 transcripts.

By pretreating simian virus 40-infected BSC-1 cells with glucosamine, [(3)H]uridine labeling of both cellular and viral RNA can be halted instantaneously by addition of cold uridine. We have studied the fate of pulse-labeled viral RNA from cells at 45 h postinfection under these conditions. During a 5-min period of labeling, both the messenger and nonmessenger regions of the late strand were transcribed. After various chase periods, nuclear viral species which sediment at 19, 17.5, and 16S were observed. Nuclear viral RNA decays in a multiphasic manner. Of the material present at the beginning of the chase period, 50% was degraded rapidly with a half-life of 8 min (initial processing). This rapidly degraded material was that fraction of the late strand which did not give rise to stable late mRNA species. Forty percent was transported to the cytoplasm, and 10% remained in the nucleus as material which sedimented in the 2 to 4S region. These 2 to 4S viral RNAs had a half-life of 3 h, and hybridization studies suggest that they are in part coded for by the late-strand nonmessenger region and are derived from the initial nuclear processing step. Another part is coded for by the late-strand messenger region and may be generated by some subsequent nuclear cleavages of 19S RNA into 17.5 and 16S RNAs. Transport of nuclear viral RNA into the cytoplasm was detected after a 5-min pulse and a 7-min chase. The maximum amount of labeled viral RNA was accumulated in the cytoplasm after a 30-min to 1-h chase. At least two viral cytoplasmic species were observed. Kinetic data suggest that 19S RNA is transported directly from the nucleus. Whether cytoplasmic 16S is formed by cleavage of 19S RNA in the cytoplasm is not clear. The half-lives of cytoplasmic 19 and 16S RNAs can be approximated as 2 and 5 h, respectively.