Modelling Spatio-Temporal Effects of Environment on Atlantic Herring, <Emphasis Type="Italic">Clupea harengus</Emphasis>

Trends in mean abundance of North Sea Atlantic herring, Clupea harengus, over the period of 1992–1995, were modelled as a function of spatial location and ocean environmental conditions using generalized additive models (GAM). In all four years, the average herring abundance was found to be highest in latitudes around 60.5°N, and decreased with increasing latitude. The thermocline depth had a significant effect on prespawning herring abundance both directly, as a main effect, and indirectly, through its interactive effect with the temperature at 60m. Average herring abundance was highest in areas having deeper thermocline depths (up to 45m) and temperatures at 60m between 9 and 11°C. Prespawning herring abundance was greater in areas of cooler surface waters in the south than in the north. Well-mixed waters and transition zones between frontal and stratified areas having sea surface temperatures mainly between 11 and 12°C and to a lesser extent between 13 and 14°C were associated with the highest herring abundance. Herring appeared to avoid the cold bottom waters in summer. Multiyear GAM analysis revealed consistent environmental preferenda of herring and affirmed further a significant decrease in herring abundance. As herring numbers declined, the population aggregated in the most preferred areas. The inter-relationships of herring and environmental factors across the study period, were similar in their structure and significance, suggesting that preferred areas for location of herring can be reasonably predicted.     

Oncology drug discovery applications using the FMAT 8100 HTS system

High-throughput screening (HTS) for potential anticancer agents requires a broad portfolio of assay platforms that may include kinase enzyme assays, protein-protein binding assays, and functional cell-based apoptosis assays. The authors have explored the use of fluorometric microvolume assay technology (the FMAT 8100 HTS System) in three distinct homogeneous HTS assays: (1). a Src tyrosine kinase enzyme assay, (2). a Grb2-SH2 protein-peptide interaction assay, and (3). an annexin V binding apoptosis assay. Data obtained from all three assays suggest that the FMAT system should facilitate the implementation of homogeneous assays for a wide variety of molecular targeted and cell-based screens.     

Delineation of the transcriptional boundaries of the lux operon of Vibrio harveyi demonstrates the presence of two new lux genes

The 5' and 3' ends of the lux mRNA of Vibrio harveyi, which extends over 8 kilobases, have been mapped, and two new genes, luxG and luxH, were identified at the 3' end of the lux operon. Both S1 nuclease and primer extension mapping demonstrated that the start site for the lux mRNA was 26 bases before the initiation codon of the first gene, luxC. The promoter region contained a typical -10 but not a recognizable -35 consensus sequence. By using S1 nuclease mapping the mRNA was found to be induced in a cell density- and arginine-dependent manner. The DNA downstream of the five known V. harveyi lux genes, luxCDABE, was sequenced and found to contain coding regions for two new genes, designated luxG and luxH, followed by a classical rho-independent termination signal for RNA polymerase. luxG codes for a protein of 233 amino acids with a molecular weight of 26,108, and luxH codes for a protein of 230 amino acids with a molecular weight of 25,326. The termination signal is active in vivo as demonstrated by 3' S1 nuclease mapping, confirming that the two genes are part of the V. harveyi lux operon. Comparison of the luxG amino acid sequence with coding regions immediately downstream from luxE in other luminescent bacteria has demonstrated that this gene may be a common component of the luminescent systems in different marine bacteria.     

A homogeneous and multiplexed immunoassay for high-throughput screening using fluorometric microvolume assay technology.

We have developed a simple, homogeneous bead-based immunoassay for use with fluorometric microvolume assay technology (FMAT). The FLISA (fluorescence-linked immunosorbent assay) can be easily adapted from existing immunoassays, is comparable to traditional ELISAs with respect to linear dynamic range and sensitivity, and can be readily performed in 96- and 384-well plates. Additionally, the FLISA utilizes 100-fold less primary antibody than the conventional immunoassay. The scanner uses a helium/neon laser to image and measure bead-bound fluorescence while the background fluorescence is ignored. Consequently, no wash steps are required to remove unbound antibody, ligand, and fluorophore. Furthermore, the instrument is capable of detecting two different fluorescent dyes, allowing for multiplexed assays based on color. Fluorescent bead-based immunoassays were developed for the cytokines IL-6 and IL-8, and their use in both one-color and two-color FLISAs is demonstrated. Although no wash steps were employed, the FLISA was able to accurately measure the concentrations of IL-6 and IL-8 in the growth media of cytokine-stimulated HUVEC cells. In addition, a simulated high-throughput two-color FLISA positively identified those wells in a 384-well plate that contained different amounts of IL-6 and/or IL-8 peptide. The homogeneous, multiplex and multiplate format of the FLISA reduces hands-on time and reagent usage, and is therefore ideally suited for high-throughput screening.     

Determination of ligand binding affinities for endogenous seven-transmembrane receptors using fluorometric microvolume assay technology.

We have developed a fluorescence-based mix and read method for the quantitative determination of receptor-ligand binding interactions. This method was used to determine IC(50) values for peptide ligands of two endogenous seven-transmembrane receptors that are expressed in cultured human cancer cells. Substance P, neurokinin A, and galanin were labeled with Cy5 and were shown to retain their native binding affinities. The cell-associated fluorescence was quantified using a fluorometric microvolume assay technology (FMAT) scanner that was designed to perform high-throughput screening assays in multiwell plates with no wash steps. The binding of fluorescently labeled substance P and neurokinin A was tested on the human astrocytoma cell line UC11 that expresses endogenous NK(1) receptor. Galanin binding was measured on endogenous galanin type 1 receptors in the Bowes neuroblastoma cell line. IC(50) values were determined for substance P, neurokinin A, and galanin and were found to correspond well with reported values from radioligand binding determinations. To demonstrate FMAT as instrumentation for high-throughput screening, it was utilized to successfully identify individual wells in a 96-well plate in which Cy5-substance P binding in UC11 cells was competed with unlabeled substance P. In addition, we developed a two-color multiplex assay in which cells individually expressing neuropeptide Y and substance P receptors were mixed in the same well. In this assay, the fluorescent ligands substance P and neuropeptide Y bound only to their respective cell types and binding was specifically competed. Therefore, two different seven-transmembrane receptor targets can be tested in one screen to minimize reagent consumption and increase throughput.     

Regulatory mutants of the Vibrio harveyi lux system

Regulatory mutants of the luminescent bacterium, Vibrio harveyi, have been isolated whose light emission can be stimulated by extracts of the growth media. Chloroform extracts of conditioned media in which V. harveyi has been grown can increase light emission in one of the dark mutants, D34, over 10³-fold. An increase in the level of the mRNA and the enzymes associated with the lux system can also be demonstrated. Analysis of the expression of the lux system in Escherichia coli transformed with DNA from the D34 regulatory mutant demonstrates that the mutation resides outside the luciferase structural genes. The results suggest that the decrease in light emission in the regulatory mutants may be due to a mutation in synthesis of an autoinducer analogous to that found for the Vibrio fischeri lux system.