A cell-based sensor system for toxicity testing using multiwavelength fluorescence spectroscopy

A novel cell-based fluorometric sensor system for toxicity monitoring is described, which uses functional spontaneously contracting cardiomyocytes (HL-1 cell line) as the biological recognition element. Based on these highly specialized cells, it has the potential of providing a sensitive and relevant analytical in vitro toxicity testing method. The system was configured by propagating the surface-attaching HL-1 cardiomyocytes in the wells of a 96-well microtiter plate and connecting the plate via an optical fiber to a fluorescence spectrometer capable of excitation-emission matrix scanning. The fluorescence data were analyzed using a conventional spectral analysis software program. The performance of the system for detection of general cytotoxicity to the cells was evaluated using three well-known drugs: verapamil, quinidine, and acetaminophen. The dose-response curves were assessed and the EC₅₀ values were determined (0.10 ± 0.007, 0.23 ± 0.025, and 12.32 ± 2.40 mM, respectively). Comparison with in vitro and in vivo reference data for the drugs showed good correlations, suggesting that this cell-based sensor system could be a useful tool in pharmacological in vitro drug testing.     

Primary inhibition: a mechanism for sudden stoppage of metachrony in ctenophores

The ctenophores Beroë cucumis and Pleurobrachia pileus exhibit rapid stoppage of comb plate beating, without accompanying muscular contraction, in response to an orally applied mechanical stimulus. This phenomenon, termed ‘primary inhibition’ by Göthlin, was re-examined by high-speed video microscopy and intracellular recording. The most remarkable features of this event were that (1) inhibition was associated with only one or two comb plates in the row, (2) inhibition occurred nearly anywhere in the ciliary beat cycle, (3) the inhibited plate acted as a mechanical blockade to the propagation of additional metachronal waves, and (4) a single depolarizing post-synaptic potential occurred nearly simultaneously with comb plate inhibition. B. cucumis and P. pileus have evolved a neurally controlled behavior to rapidly stop comb plate metachrony.

     

Body shape variation in the Characid Psalidodon rivularis from São Francisco river,Southeast Brazil (Characiformes: Stethaprioninae)

Psalidodon rivularis is a species of tetra endemic to the São Francisco River basin and, based on cytogenetic and molecular studies, represents a complex of species. The objective of the present work was to identify morphological differences in the body shape of seven populations of P. rivularis from the Upper and Middle São Francisco River basin through geometric morphometry. In all, we photographed 174 individuals on the right side of the body and 17 landmarks were digitized on each image. To study the effects of allometry on the shape, we performed regression analysis and, to study shape modulation at different collection points and sub-basins, the canonical variation analyses. We found differences in shape between collection points and sub-basins associated with relative body height and sub-orbital plate recoil, in addition to a significant influence of size on specimen shape (allometry) associated with the ventral skull, orbits and sub-orbital plate. We do not envision differences in body shape between males and females. Several works with fish relate body height with water velocity, while the sub-orbital plate recoil shows a taxonomic or ecological potential, marking the main difference between the populations of the Upper and Middle São Francisco River.     

Effect of brefeldin A and the dynamics of the actin plate on cytokinesis of zygotes in the brown alga,Silvetia babingtonii (Fucales,Phaeophyceae)

In the cytokinesis of brown algae, actin filaments appear like a plate at the intersecting region of microtubules (MTs) that emerge from the centrosomes after mitosis. The function of the actin plate itself is still unknown. To elucidate the relationship between the actin plate, MTs and membrane fusion, without inducing cytoskeleton depolymerization, the effect of brefeldin A (BFA), which prevents the production of vesicles from Golgi bodies, was examined in zygotes of Silvetia babingtonii. The beginning of mitosis was slightly delayed in zygotes under BFA compared with the controls. Almost all zygotes were inhibited for the progression of cytokinesis by BFA treatment. Ultrastructural observations showed that Golgi cisternae became fragmented or curled following continuous treatment with BFA, and the inhibitory status of cytokinesis between zygotes. The next cell cycle started before cytokinesis was completed. Although the appearance of the actin plate was not disturbed by BFA treatment, the behaviour of the actin plate during the transition between the first and second cell cycles could be classified into two patterns: it was either invisible upon the initiation of the next cell cycle, or a portion of it remained even though the next cell cycle had begun. In the latter case, a part of the actin plate seemed to associate with the new partially formed cell partition membrane, and MTs from the centrosomes were bound to it. The actin plate completely disappeared in the next mitosis, then re-emerged in the middle area of the four daughter nuclei. The results of the present study indicated that, under BFA treatment, the actin plate persisted until just before the beginning of the next mitotic phase, when the new, incomplete cell partition membrane was present, and MTs sustained the actin plate until next mitosis.     

An in vitro experimental study on the relationship between pulsatile tinnitus and the dehiscence/thinness of sigmoid sinus cortical plate

Pulsatile tinnitus (PT), characterized as pulse-synchronous, is generally objective. Sigmoid sinus (SS) venous sound is widely suggested to be a possible sound source of PT. The dehiscence and thinness of SS cortical plate (CP) was commonly reported as PT pathology in previous studies, but lack quantitative or biomechanical analysis. In this study, it was aimed to quantify the relationship between venous sound and CP dehiscence/thinness using in vitro experiment. The in vitro models of SS and CP were established based on 3D-printing, with the developed pulsatile venous flow in the SS model. The generated sound signal and the vibration response at the dehiscent/thinned area were analyzed. The sound signal generated in the normal-sized dehiscence model was pulse-synchronous within 100-–400 Hz, which had similar acoustic characteristics as the clinical PT sounds. It was concluded that the pulsatile venous sound is produced at TS-SS junction in case of CP dehiscence. The CP, even a thinned one can effectively diminish the venous sound and sound-generating pulsatile vibration at TS-SS junction. The CP dehiscence would induce pulse-synchronous and high pressure venous sound, as well as pulse-synchronous vibration above 20 Hz, regardless of the dehiscence size. On the contrary, the CP thinness would not induce obvious venous sound or pulsatile vibration above 20 Hz.     

Induction of multinucleation by β-glucosyl Yariv reagent in regenerated cells from <Emphasis Type="Italic">Marchantia polymorpha</Emphasis> protoplasts and involvement of arabinogalactan proteins in cell plate formation

Arabinogalactan proteins (AGPs) are abundant plant cell surface proteoglycans widely distributed in plant species. Since high concentrations of β-glucosyl Yariv reagent (βglcY), which binds selectively to AGPs, inhibited cell division of protoplast-regenerated cells of the liverwort Marchantia polymorpha L. (Shibaya and Sugawara in Physiol Plant 130:271–279, 2007), we investigated the mechanism underlying the inability of the cells to divide normally by staining nuclei, cell walls and β-1,3-glucan. Microscopic observation showed that the diameter of regenerated cells cultured with βglcY was about 2.8-fold larger than that of cells cultured without βglcY. The cells cultured with βglcY were remarkably multinucleated. These results indicated that βglcY did not inhibit mitosis but induced multinucleation. In the regenerated cells cultured with low concentrations of βglcY (5 and 1 μg ml⁻¹), the cell plate was stained strongly by βglcY, suggesting abundant AGPs in the forming cell plate. In these cell plates, β-1,3-glucan was barely detectable or not detected. In multinucleated cells, cell plate-like fragments, which could not reach the cell wall, were frequently observed and they were also stained strongly by βglcY. Our results indicated that AGPs might have an important role in cell plate formation, and perturbation of AGPs with βglcY might result in remarkable multinucleation in protoplast-regenerated cells of M. polymorpha. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

End plate classification and spontaneous sprouting in the sternocostalis muscle of the rat: A new whole mount preparation

Summary The sternocostalis muscle of the rat provides an ideal preparation for examination of neuromuscular junctions in a whole mount. It contains all three morphological end plate types (A, B and C), and its segmental innervation allows ease of experiment, such as partial denervation.The ratio of end plate types remains constant in all individuals of the same age, and there is no variation in this ratio over different regions of the same muscle. Spontaneous sprouting was observed from end plates of all the animals examined: again the ratio of sprouting end plate types remained constant over all muscles examined, and over all regions of the same muscle.     

Expression of the mouse PR domain protein Prdm8 in the developing central nervous system

It was first shown in the PR (PRDI-BF1 and RIZ homology) domain family proteins that the PR domain has homology to the SET (Su(var)3-9, Enhancer-of-zeste and Trithorax) domain, a catalytic domain of the histone lysine methyltransferases. Recently, there are many reports that the PR domain proteins have important roles in development and/or cell differentiation. In this report, we show the expression patterns of one of the mouse PR domain proteins, Prdm8, in the developing central nervous system. In the developing retina, Prdm8 expression was detected in postmitotic neurons in the inner nuclear layer and the ganglion cell layer, and its expression became restricted predominantly to the rod bipolar cells when retinogenesis was completed. In the developing spinal cord, Prdm8 was expressed first in the progenitor populations of ventral interneurons and motor neurons, and later in a subpopulation of interneurons. In the developing brain, Prdm8 expression was observed in postmitotic neurons in the intermediate zone and the cortical plate. In the postnatal brain, Prdm8 was expressed mainly in layer 4 neurons of the cerebral cortex. These results show that Prdm8 expression is tightly regulated in a spatio-temporal manner during neural development and mainly restricted to postmitotic neurons, except in the spinal cord.     

Mesodermal patterning during avian gastrulation and neurulation: Experimental induction of notochord from non-notochordal precursor cells

The cells that are normally fated to form notochord occupy a region at the rostral tip of the primitive streak at late gastrula/early neurula stages of avian and mammalian development. If these cells are surgically removed from avian embryos in culture, a notochord will nonetheless form in the majority of cases. The origin of this reconstituted notochord previously had not been investigated and was the objective of this study. Chick embryos at late gastrulal early neurula stages were cultured, and the rostral tip of the primitive streak including Hensen's node was removed and replaced with non-node cells from quail epiblast to ensure that the cells normally fated to be notochord would be absent and that healing of the blastoderm would occur. Embryos were allowed to develop for 24 hr, and the presence and origin (host or graft) of the notochord were assessed using antibodies against notochord or quail cells. Two notochords typically developed; both were almost exclusively of host origin. The primitive streak, and in some cases adjacent tissues, was removed from another group of embryos in an attempt to estimate the mediolateral position and extent of the cells required to form reconstituted notochord. Additional experimental embryos with and without grafts were transected at various rostrocaudal levels in an attempt to estimate the rostrocaudal extent of the cells required to form reconstituted notochord. Finally, various levels of the primitive streak either were placed in a neutral environment (the germ cell crescent) or were grafted in place of the node. Collective results from all experiments indicate that the areas lateral to the rostral portion of the primitive streak, estimated to have a rostrocaudal span of less than 500 μm and a mediolateral extent of less than 250 μm, are critical for formation of the reconstituted notochord. Fate mapping and histological examination of this region identify 4 possible precursor cell populations. Further studies are underway to determine which of the 4 possible precursor cell types forms or induces the reconstituted notochord, and which tissue interactions underlie this change in cell fate. © 1995 Wiley-Liss, Inc.