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.     

Glycoconjugate with terminal galactose. A selective property of macrophages in developing rat lung

Pulmonary macrophages in pre- and postnatal rats were examined histochemically with a battery of peroxidase labeled lectins. Among them, Griffonia simplicifolia agglutinin I-B4 (GSA I-B4) which binds specifically to terminal alpha-galactose showed selective affinity in lung for the monocyte-macrophage line. These cells were demonstrable with GSA I-B4 from the 14th day of gestation through the adult. Extension to the ultrastructural level showed strong selective binding of this lectin to the surface of the plasmalemma and inner face of membranes limiting phagosomes in macrophages. At day 14 of gestation, monocyte-like cells positive with GSA I-B4 were scattered in various organs including lung. The lectin reactive cells in lung increased in number and size with development, infiltrating the interstitium through day 20 of gestation and then also entering the alveolar space. These findings suggest that GSA I-B4 recognizes a surface glycoconjugate characteristic of the pulmonary monocyte-macrophage line. Such selective lectin affinity offers a marker for detecting the pulmonary macrophages and examining their kinetics by light and electron microscopy.     

Histochemical evaluation of glycoconjugates in the male reproductive tract with lectin-horseradish peroxidase conjugates: I. Staining of principal cells and spermatozoa in the mouse

Several glycoconjugates are thought to bind spermatozoa as they pass through reproductive ducts. Paraffin sections of testis, ductuli efferentes, epididymis, and vas deferens of male mice were stained with ten different lectin-horseradish peroxidase conjugates to localize possible sites of synthesis and secretion of such glycoconjugates, based on the carbohydrate moieties in their constituent oligosaccharide side chains. Principal (columnar) cells lining the efferent ducts, germinal epithelium, and developing and maturing spermatozoa were examined with light microscopy. Staining of the Golgi and apical zones of cells was interpreted as evidence for synthesis and secretion of glycoconjugates. Principal cells synthesized and secreted glycoconjugates with sugar moieties as follows: sialic acid, all regions of the efferent ducts examined; the terminal disaccharide D-galactose- (beta 1----3) -N-acetyl-D-galactosamine, all regions of ducts except epididymis I; terminal alpha-D-galactosamine, some cells in epididymis III-V; N-acetyl-D-galactosamine, ductuli efferentes, epididymis I, II, and some cells in epididymis III-V; alpha-L-fucose, ductuli efferentes, vas deferens, and all regions of the epididymis except IV; N-glycosidic side chains, ductuli efferentes, vas deferens, and epididymis I, IV, and V. All of these sugar residues as well as N-acetyl-D-glucosamine were associated with the acrosomes and tails of spermatozoa throughout the ducts except for alpha-N-acetyl-D-galactosamine in epididymis I, and all occurred during one or more stages of spermiogenesis. The synthesis and secretion of glycoconjugates that bind to spermatozoa appear to involve more regions of the primary reproductive structures than was believed previously.     

Expression of binding sites for Dolichos biflorus agglutinin at the apical aspect of collecting duct cells in rat kidney

Summary To identify precisely the structural and functional cell type in the collecting duct of the rat kidney expressing binding sites for Dolichos biflorus agglutinin (DBA), we stained serial paraffin sections of kidney with horseradish peroxidase-labeled DBA and with immunocytochemical methods for localizing (Na⁺+K⁺)-ATPase and carbonic anhydrase II (CA II), enzymes found preferentially in principal and intercalated cells, respectively. Most principal cells expressing a strong basolateral staining for (Na⁺ + K⁺)-ATPase showed binding sites for DBA at their luminal surfaces. However, a minority of cells rich in CA II and showing morphologic characteristics of intercalated cells also expressed DBA binding sites at their luminal surface and apical cytoplasm. These data suggest that DBA cytochemistrycan provide a useful tool for studying the functional polarity of the main cell types of the collecting duct of the rat kidney.     

Mechanism of action of 2-hydroxyestradiol on steroidogenesis in ovarian granulosa cells: interactions with catecholamines and gonadotropins involve cyclic adenosine monophosphate

Previously we have shown that 2-hydroxyestradiol (2-OH-E2) synergizes with catecholamines to enhance progesterone production by porcine granulosa cells in vitro. The present studies were undertaken to determine if the synergistic effects of 2-OH-E2 and catecholamines were 1) modulated by gonadotropins, 2) unique to catecholamines, and 3) mediated by cyclic adenosine monophosphate (cAMP). Undifferentiated granulosa cells from 1- to 3-mm porcine follicles were cultured in serum-free medium for periods of 6-9 days. A 3-day pretreatment plus a 4-day cotreatment versus a 4-day cotreatment of granulosa cell cultures with follicle-stimulating hormone (FSH) did not significantly alter progesterone production stimulated by a saturating concentration of epinephrine (EPI; 2 micrograms/ml) but significantly reduced the effect of 4 micrograms/ml 2-OH-E2 on Day 7 of culture. Four-day cotreatment of either FSH or luteinizing hormone (LH) from Day 3 to 7 of culture dramatically enhanced progesterone production stimulated by 2-OH-E2 and estradiol (E2) but not by EPI when measured on Day 7 of culture. Progesterone production (expressed as "-fold of controls") stimulated by 4-day treatment of EPI, 2-OH-E2, or EPI-plus-2-OH-E2 was 1.4 +/- 0.2, 8.2 +/- 2.2, and 10.7 +/- 1.0, respectively, in the presence of LH (n = 5 experiments), and 1.9 +/- 0.1, 7.8 +/- 1.4, and 10.6 +/- 1.8, respectively, in the presence of FSH (n = 3 experiments). Similar to E2, 2-OH-E2 significantly enhanced the stimulating effect of the cAMP analog 8-bromo-cAMP (0.5 mM) on progesterone production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of glycoconjugates at the surface of developing type II pneumocytes and Clara cells

Summary The apical surface coat of type II pneumocytes and Clara cells in pre- and post-natal rat lung was examined with lectin histochemical methods. Lectins fromHelix pomatia (HPA), peanut (PNA) andMaclura pomifera (MPA) were conjugated with horseradish peroxidase and used to stain paraffin sections of fixed lung with or without certain pre-treatments. HPA and MPA were observed to react with almost all type II pneumocytes at postnatal day 1. Type II pneumocytes that stained with a sialidase—PNA sequence increased from a few positive cells at postnatal day 5 to many in the adult. It has been reported that the surface coat of type II pneumocytes closely resembles that of Clara cells in its staining with histochemical methods employing cationic dyes or lectins including MPA and PNA. However, staining with HPA, especially after periodic acid oxidation, revealed many type II pneumocytes with strong reactivity but showed only a few Clara cells that were faintly positive. HPA also stained alveolar macrophages. The HPA affinity of macrophages, however, was labile to oxidation with periodic acid or galactose oxidase unlike that of type II pneumocytes. This difference suggests that HPA recognizes more than one type of sugar structure.To whom all correspondence and reprint requests should be addressed.     

Molecular evolution among someDrosophila species groups as indicated by two-dimensional electrophoresis

Summary The evolutionary and phylogenetic relationships of sevenDrosophila species groups (represented byD. melanogaster, D. mulleri, D. mercatorum, D. robusta, D. virilis, D. immigrans, D. funebris, andD. melanica) were investigated by the use of two-dimensional electrophoresis. The resulting phylogeny is congruent with the current views of evolution among these groups based on morphological characters and immunological distances. Previous studies indicated that the ability of one-dimensional electrophoresis to resolve relationships between distantly related taxa extended to about the Miocene [25 million years (Myr) ago], but the present study demonstrates that two-dimensional electrophoresis is a useful indicator of phylogeny even back to the Paleocene (65 Myr ago). In addition, two-dimensional electrophoresis is shown to be a useful technique for detecting slowly evolving structural proteins such as actins and tropomyosins.     

Immunohistochemical localization of Na+,K+-ATPase in rodent and human salivary and lacrimal glands

The enzyme Na+,K+-ATPase was localized immunohistochemically in major salivary glands of mouse, rat, and human and in exorbital lacrimal glands of the rodents. Immunoreactive Na+,K+-ATPase was abundant in the basolateral membranes of all epithelial cells lining striated and intra- and interlobular ducts of all glands. Reactivity of intercalated ducts varied among gland type and species. Cells lining granular ducts in rodent submandibular gland showed a heterogeneous staining pattern in rat but stained homogeneously in mouse. Secretory cells varied greatly in their content of immunoreactive Na+,K+-ATPase. As with all duct cells, staining was present only at the basolateral surface and was never observed at the luminal surface of reactive secretory cells. Mucous cells failed to show any reactivity in any gland examined. Serous cells showed a gradient of immunostaining intensity ranging from strongly positive in demilunes of human sublingual gland to negative in rat submandibular gland and lacrimal glands of rats and mice. The presence of basolaterally localized Na+,K+-ATPase in most serous cells but not in mucous cells suggests that the enzyme contributes to the ion and water content of copious, low-protein serous secretions. The intense immunostaining of cells in most if not all segments of the duct system supports the idea that the ducts are involved with modification of the primary saliva, and extends this concept to include all segments of the duct system.     

Variability in vibration perception threshold among sites: a potential source of error in biothesiometry

Vibration perception threshold was measured with a biothesiometer by a single observer at both medial malleoli and both big toes in 110 diabetic patients aged 15-65 selected at random and in 64 non-diabetic subjects aged 20-65. The vibration perception threshold showed appreciable individual variation both between contralateral sites and between ipsilateral sites, differing by at least 30% between the big toes in 26 (24%) of the diabetic patients and 16 (25%) of the non-diabetic group. Variability between sites was significantly greater in the diabetics than the normal subjects. The vibration perception threshold exceeded published normal values at one or more sites in 22 of the diabetic patients but at all four sites in only four.The wide variability in vibration perception threshold among sites may be due to the tissue characteristics locally and, in diabetic patients, possibly to asymmetric neuropathy. Biothesiometer readings at single or unilateral sites may be unrepresentative or misleading.