Combining Motion Analysis and Microfluidics – A Novel Approach for Detecting Whole-Animal Responses to Test Substances

Small, early life stages, such as zebrafish embryos are increasingly used to assess the biological effects of chemical compounds in vivo. However, behavioural screens of such organisms are challenging in terms of both data collection (culture techniques, drug delivery and imaging) and data evaluation (very large data sets), restricting the use of high throughput systems compared to in vitro assays. Here, we combine the use of a microfluidic flow-through culture system, or BioWell plate, with a novel motion analysis technique, (sparse optic flow - SOF) followed by spectral analysis (discrete Fourier transformation - DFT), as a first step towards automating data extraction and analysis for such screenings. Replicate zebrafish embryos housed in a BioWell plate within a custom-built imaging system were subject to a chemical exposure (1.5% ethanol). Embryo movement was videoed before (30 min), during (60 min) and after (60 min) exposure and SOF was then used to extract data on movement (angles of rotation and angular changes to the centre of mass of embryos). DFT was subsequently used to quantify the movement patterns exhibited during these periods and Multidimensional Scaling and ANOSIM were used to test for differences. Motion analysis revealed that zebrafish had significantly altered movements during both the second half of the alcohol exposure period and also the second half of the recovery period compared to their pre-treatment movements. Manual quantification of tail flicking revealed the same differences between exposure-periods as detected using the automated approach. However, the automated approach also incorporates other movements visible in the organism such as blood flow and heart beat, and has greater power to discern environmentally-driven changes in the behaviour and physiology of organisms. We suggest that combining these technologies could provide a highly efficient, high throughput assay, for assessing whole embryo responses to various drugs and chemicals.     

A study of cytoplasmic and nuclear estrogen and progestin receptors in gynecologic neoplasms

A rapid method for simultaneous preparation of cytosol and nuclear estrogen (E) and progestin (P) receptors and their in vitro determination is described. The method was applied to several uterine or ovarian surgical specimens to evaluate their steroid hormone "dependence". The results suggest that low cytoplasmic E receptor levels (ERc) are associated with higher nuclear E receptor (ERn) levels but no apparent correlation was observed between PRc and ERn levels. The method appeared to be suitable for screening steroid hormone receptor content in tumor tissues and may provide better estimation of steroid dependence since both cytoplasmic and nuclear compartments can be studied simultaneously.     

Regulation of expression of a sheep metallothionein 1a-sheep growth hormone fusion gene in transgenic mice.

Transgenic mice containing a sheep metallothionein 1a-sheep growth hormone fusion gene exhibited low, tissue-specific basal levels of transgene mRNA expression, resulting in slightly elevated levels of circulating growth hormone that did not lead to a detectable increase in growth. After zinc stimulation, high levels of transgene mRNA expression were induced in a number of tissues; these levels correlated with increased levels of circulating growth hormone, resulting in growth increases of up to 1.5 times the levels of controls and unstimulated transgenic mice. After removal of the zinc stimulus, transgene expression and circulating growth hormone concentrations returned to basal levels. Additional evidence from the pattern of developmental expression of the transgene suggests that zinc is the main regulator of this promoter in mice. The demonstrated regulation and low basal level of expression of the sheep metallothionein 1a promoter make it a candidate for use in other mouse transgenic studies and for use in transgenic livestock, in which regulation of expression is essential.     

Identification of the glucose transporter in mammalian cell membranes with a 125I-forskolin photoaffinity label.

The glucose transporter has been identified in a variety of mammalian cell membranes using a photoactivatable carrier-free radioiodinated derivative of forskolin, 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n ([125I]IAPS-forskolin) at 1-3 nM. The membranes that were photolabelled with [125I]IAPS-forskolin were human placental membranes, rat cortical and cerebellar synaptic membranes, rat cardiac sarcolemmal membranes, rat adipocyte plasma membranes, smooth-muscle membranes, and S49 wild-type (WT) lymphoma-cell membranes. The glucose transporter in plasma membranes prepared from the insulin-responsive rat cardiac sarcolemmal cells, rat adipocytes and smooth-muscle cells were determined to be approx. 45 kDa by SDS/polyacrylamide-gel electrophoresis (PAGE). Photolysis of human placental membranes, rat cortical and cerebellar synaptic membranes, and WT lymphoma membranes with [125I]-IAPS-forskolin, followed by SDS/PAGE, indicated specific derivatization of a broad band (43-55 kDa) in placental membranes and a narrower band (approx. 45 kDa) in synaptic membranes and WT lymphoma membranes. Digestion of the [125I]IAPS-forskolin-labelled placental and WT lymphoma membranes with endo-beta-galactosidase showed a reduction in the apparent molecular mass of the radiolabelled band to approx. 40 kDa. The membranes that were photolabelled with [125I]IAPS-forskolin and trypsin-treated produced a radiolabelled proteolytic fragment with an apparent molecular mass of 18 kDa. [125I]IAPS-forskolin is a highly effective probe for identifying low levels of glucose transporters in mammalian tissues.     

Regulation of γ-Aminobutyric Acid/Barbiturate Receptor-Gated Chloride Ion Flux in Brain Vesicles by Phospholipase A2: Possible Role of Oxygen Radicals

Preincubation of brain membranes with phospholipase A2 (PLA2) has been shown previously to affect the binding characteristics of various recognition sites associated with the gamma-aminobutyric acid (GABA) receptor complex. In the present study, we have investigated the effects of PLA2 (from Naja naja siamensis venom) on the functional activity of the GABA receptor/chloride ion channel. PLA2 (0.001-0.02 U/mg protein) preincubation decreased pentobarbital-induced 36Cl- efflux and muscimol-induced 36Cl- uptake in rat cerebral cortical synaptoneurosomes. The effect of PLA2 was prevented by EGTA and two nonselective PLA2 inhibitors, mepacrine and bromophenacyl bromide. The removal of free fatty acids by addition of bovine serum albumin both prevented and reversed the effect of PLA2. Products of the catalytic activity of PLA2, such as the unsaturated free fatty acids, arachidonic and oleic acids, mimicked the effect of PLA2. However, the saturated fatty acid, palmitic acid, and lysophosphatidyl choline had no effect on pentobarbital-induced 36Cl- efflux. Because unsaturated free fatty acids are highly susceptible to peroxidation by oxygen radicals, the role of oxygen radicals was investigated. Xanthine plus xanthine oxidase, a superoxide radical generating system, mimicked the effect of PLA2, whereas the superoxide radical scavenger, superoxide dismutase, diminished the effects of PLA2 and arachidonic acid on pentobarbital-induced 36Cl- efflux. Similarly, the effect of PLA2 was also inhibited by methanol (1 mM), a scavenger of the hydroxyl radical, and by catalase. These data indicate that exogenously added PLA2 induces alterations in membrane phospholipids, possibly promoting the generation of oxygen radicals and fatty acid peroxides which can ultimately modulate GABA/barbiturate receptor function in brain.     

Differences in the Biophysical Properties of the Benzodiazepine/γ-Aminobutyric Acid Receptor Chloride Channel Complex in the Long-Sleep and Short-Sleep Mouse Lines

Significant differences in the thermal stability of benzodiazepine receptors were found in cerebral cortical membranes prepared from the long-sleep (LS) and short-sleep (SS) selected mouse lines. Thus, benzodiazepine receptors from LS mice were heat inactivated (55 degrees C) at a significantly faster rate than those from SS mice. Although gamma-aminobutyric acid (GABA) reduced the rate of heat inactivation in both lines, the more rapid rate of inactivation in the LS line was maintained. Furthermore, the potency of GABA to enhance [3H]flunitrazepam binding decreased threefold in membranes from LS mice as the incubation temperature was increased from 0 degrees to 37 degrees C, but was unaltered in membranes from SS mice. These differences in the biophysical properties of the benzodiazepine/GABA receptor chloride channel complex ("supramolecular complex"), together with a higher KD for t-[35S]butylbicyclophosphorothionate in membranes from LS compared to SS mice, suggest that the supramolecular complex may modulate the differential sensitivity to some depressants and convulsants in these lines.     

p53 is frequently mutated in Burkitt''s lymphoma cell lines.

A panel of 12 Burkitt's lymphoma cell lines and four other B cell lines were tested for the presence of mutations in p53. Protein analysis using a mutant-specific antibody and sequencing of both cDNA and genomic DNA revealed changes relative to the standard p53 protein sequence in 12 of the 16 lines studied, including 10 of the BL lines. Mutation of p53 in the BL lines was usually accompanied by loss of the other allele of p53. Testing of the mutated p53 cDNAs for gain of transforming activity or loss of growth suppression activity showed that several of the BL mutants were functionally altered from wild-type p53.     

Genetic Controls over Activities of Tyrosinase and Dopachrome Conversion Factor in Murine Melanocytes

We evaluated the three catalytic activities of tyrosinase and one activity of dopachrome conversion factor (DCF) in extracts made from skins of 6-day-old yellow and nonyellow mice. At least one of the catalytic activities of tyrosinase and of DCF correlate with the color of pigment being produced in the hair follicles of the mice. We use these data to evaluate existing hypotheses about the mechanism of the interacting genetic controls over melanogenesis.     

Subcellular Localization of "Peripheral-Type" Binding Sites for Benzodiazepines in Rat Brain

The binding of [3H]Ro 5-4864, a specific ligand for "peripheral-type" benzodiazepine binding sites and [3H]Ro 15-1788, a specific ligand for the central benzodiazepine receptors, was determined in subcellular fractions of rat brain. As previously reported, the highest levels of "peripheral-type" benzodiazepine binding sites and benzodiazepine receptors were found in the crude P1 and P2 fractions, respectively. Purification of these crude fractions revealed that high levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding were present in the mitochondrial and synaptosomal fractions. In contrast, the purified nuclei and myelin contained low levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding.     

GABAA Receptor Complex in an Experimental Model of Hepatic Encephalopathy: Evidence for Elevated Levels of an Endogenous Benzodiazepine Receptor Ligand

The involvement of the gamma-aminobutyric acidA (GABAA) receptor complex in the pathogenesis of hepatic encephalopathy was examined in thioacetamide-treated rats with fulminant hepatic failure. Partially purified extracts from encephalopathic rat brain were approximately three times more potent in inhibiting [3H]Ro 15-1788 binding to benzodiazepine receptors than identically prepared extracts from control rats. High levels of inhibitory activity were also found in extracts of plasma, heart, and liver from thioacetamide-treated rats. The inhibition of [3H]Ro 15-1788 binding by brain extracts appeared to be competitive and reversible and was unaffected by treatment with either proteolytic enzymes or boiling. Further, GABA significantly enhanced the potency of these extracts in inhibiting [3H]flunitrazepam binding. In contrast, no differences were found in radioligand binding to the constituent recognition sites of the GABAA receptor complex in well-washed brain membranes prepared from control and encephalopathic animals. These findings suggest that the recognition-site qualities of the constituent proteins of the GABAA receptor complex are unchanged in an experimental model of hepatic encephalopathy. However, significant elevations in the level of a substance or substances with neurochemical properties characteristic of a benzodiazepine receptor agonist may contribute to the electrophysiological and behavioral manifestations of hepatic encephalopathy.