Hydrodynamics of the fast-start caridoid escape response in Antarctic krill, Euphausia superba.

Krill are shrimp-like crustaceans with a high degree of mobility and variety of documented swimming behaviors. The caridoid escape response, a fast-start mechanism unique to crustaceans, occurs when the animal performs a series of rapid abdominal flexions and tail flipping that results in powerful backward strokes. The current results quantify the animal kinematics and three-dimensional flow field around a free-swimming Euphausia superba as it performs the caridoid escape maneuver. The specimen performs a single abdominal flexion-tail flip combination that leads to an acceleration over a 42 ms interval allowing it to reach a maximum speed of 57.0 cm/s (17.3 body lengths/s). The krill's tail flipping during the abdominal closure is a significant contributor to the thrust generation during the maneuver. The krill sheds a complex chain of vortex rings in its wake due to the viscous flow effects while the organism accelerates. The vortex ring structure reveals a strong suction flow in the wake, which suggests that the pressure distribution and form drag play a role in the force balance for this maneuver. Antarctic krill typically swim in a low to intermediate Reynolds number (Re) regime where viscous forces are significant, but as shown by this analysis, its high maneuverability allows it to quickly change its body angle and swimming speed.

Copepods' Response to Burgers' Vortex: Deconstructing Interactions of Copepods with Turbulence.

This study examined the behavioral response of two marine copepods, Acartia tonsa and Temora longicornis, to a Burgers' vortex intended to mimic the characteristics of a turbulent vortex that a copepod is likely to encounter in the coastal or near-surface zone. Behavioral assays of copepods were conducted for two vortices that correspond to turbulent conditions with mean dissipation rates of turbulence of 0.009 and 0.096 cm(2) s(-3) (denoted turbulence level 2 and level 3, respectively). In particular, the Burgers' vortex parameters (i.e., circulation and rate of axial strain rate) were specified to match a vortex corresponding to the median rate of dissipation due to viscosity for each target level of turbulence. Three-dimensional trajectories were quantified for analysis of swimming kinematics and response to hydrodynamic cues. Acartia tonsa did not significantly respond to the vortex corresponding to turbulence level 2. In contrast, A. tonsa significantly altered their swimming behavior in the turbulence-level-3 vortex, including increased relative speed of swimming, angle of alignment of the trajectory with the axis of the vortex, ratio of net-to-gross displacement, and acceleration during escape, along with decreased turn frequency (relative to stagnant control conditions). Further, the location of A. tonsa escapes was preferentially in the core of the stronger vortex, indicating that the hydrodynamic cue triggering the distinctive escape behavior was vorticity. In contrast, T. longicornis did not reveal a behavioral response to either the turbulence level 2 or the level 3 vortex.

Bioinspired algorithm for autonomous sensor-driven guidance in turbulent chemical plumes.

We designed and implemented a control algorithm for sensor-mediated chemical plume tracking in a turbulent flow environment. In our design, we focused on development of a signal processing strategy capable of replicating behavioral responses of actively tracking blue crabs (Callinectes sapidus) to chemical stimuli. The control algorithm is evaluated in a hardware platform that allows motion in two directions (i.e. forward-back and left-right). The geometric arrangement of the sensor array is inspired by the location of blue crab sensor populations. Upstream motion is induced by a binary response to supra-threshold spikes of concentration, and cross-stream steering is controlled by contrast between bilaterally-separated sensors. Like animal strategies, the developed control algorithm is dynamic. This property allows the algorithm to function effectively in the highly irregular turbulent environment and produces adaptive adjustments of motion to minimize the distance to the source of a plume. Tracking trials indicate that roughly 80% of the tracks successfully stop near the plume source location. Both success rate and movement patterns of the tracker compare favorably to that of blue crabs searching for odorant plume sources, thus suggesting that our sensory-mediated behavior hypothesis are generally accurate and that the associated tracking mechanisms may be successfully implemented in hardware.

Cyclin-C-dependent cell-cycle entry is required for activation of non-homologous end joining DNA repair in postmitotic neurons.

It is commonly believed that neurons remain in G(0) phase of the cell cycle indefinitely. Cell-cycle re-entry, however, is known to contribute to neuronal apoptosis. Moreover, recent evidence demonstrates the expression of cell-cycle proteins in differentiated neurons under physiological conditions. The functional roles of such expression remain unclear. Since DNA repair is generally attenuated by differentiation in most cell types, the cell-cycle-associated events in postmitotic cells may reflect the need to re-enter the cell cycle to activate DNA repair. We show that cyclin-C-directed, pRb-dependent G(0) exit activates the non-homologous end joining pathway of DNA repair (NHEJ) in postmitotic neurons. Using RNA interference, we found that abrogation of cyclin-C-mediated exit from G(0) compromised DNA repair but did not initiate apoptosis. Forced G(1) entry combined with prevention of G(1) --> S progression triggered NHEJ activation even in the absence of DNA lesions, but did not induce apoptosis in contrast to unrestricted progression through G(1) --> S. We conclude that G(0) --> G(1) transition is functionally significant for NHEJ repair in postmitotic neurons. These findings reveal the importance of cell-cycle activation for controlling both DNA repair and apoptosis in postmitotic neurons, and underline the particular role of G(1) --> S progression in apoptotic signaling, providing new insights into the mechanisms of DNA damage response (DDR) in postmitotic neurons.

Use of the newborn screening card to define cause of death in a 12-year-old diagnosed with epilepsy.

Post-mortem investigation of sudden death in young people frequently reveals no overt cause for the death. Full investigation is hampered if tissue or blood is not retained for DNA analysis. We report a post mortem molecular diagnosis of long QT syndrome in a 12-year-old boy diagnosed with epilepsy who died suddenly playing sport. The DNA was extracted from an archived blood spot on his newborn screening ('Guthrie') card, which had been taken from him at 6 days of age. A missense mutation was detected in exon 5 of the KCNQ1 gene; R243C (835C > T), associated with long QT type 1. The same mutation was found in the mother (who now takes effective preventative therapy), but not in the sib who has now been reassured that she is not at risk of sudden death.

Fluid mechanics produces conflicting, constraints during olfactory navigation of blue crabs, Callinectes sapidus.

Foraging blue crabs must respond to fluid forces imposed on their body while acquiring useful chemical signals from turbulent odor plumes. This study examines how blue crabs manage these simultaneous demands. The drag force, and hence the cost of locomotion, experienced by blue crabs is shown to be a function of the body orientation angle relative to the flow. Rather than adopting a fixed orientation that minimizes the drag, blue crabs decrease their relative angle (increase drag) when odor is present in low speed flow, while assuming a drag-minimizing posture under other conditions. The motivation for crabs to adopt an orientation with larger drag appears to relate to their ability to acquire chemical signal information for odor tracking. In particular, when orienting at a smaller angle relative to the flow direction, more concentrated odor filaments arrive at the antennules to mediate upstream movement, allowing a more useful bilateral comparison between the appendage chemosensors to be made. Blue crabs respond to conflicting demands by weighting the degree of drag minimization in proportion to the potential magnitude of the drag cost and the potential benefit of acquiring chemosensory cues. Higher flow velocity magnifies the locomotory cost of a high drag posture, thus in swift flows crabs minimize drag and sacrifice their ability to acquire olfactory cues.

Chemical plume tracking. 1. Chemical information encoding.

It is shown experimentally that chemical information can be encoded and preserved in flowing liquid streams. It can be retrieved by chemical sensing arrays using correlation analysis. This finding is important for understanding of the mechanism of chemotaxis as practiced by some aquatic animals and also is a necessary prerequisite for construction of chemical plume tracking robots.

Localized injury in cardiomyocyte network: a new experimental model of ischemia-reperfusion arrhythmias.

We developed a new experimental approach to study the effects of local injury in a multicellular preparation and tested the ability of the method to induce reperfusion arrhythmias in cardiomyocyte monolayers. A small region of injury was created using geometrically defined flows of control and ischemia-like solutions. Calcium transients were acquired simultaneously from injured, control, and border zone cells using fluo 4. Superfusion with the injury solution rapidly diminished the amplitude of calcium transients within the injury zone, followed by cessation of cell beating. Reperfusion caused an immediate tachyarrhythmic response in approximately 17% of experiments, with a wave front propagating from a single cell or small cell cluster within the former injury zone. Inclusion of a gap junction uncoupler (1 mM heptanol) in the injury solution narrowed the functional border and sharply increased the number of ectopic foci and the incidence of reperfusion arrhythmias. The model holds a potential to reveal both micro- and macroscopic features of propagation, conduction, and cell coupling in the normal and diseased myocardium and to serve as a new tool to test antiarrhythmic protocols in vitro.

Dimethoate inhibits steroidogenesis by disrupting transcription of the steroidogenic acute regulatory (StAR) gene.

Dimethoate is a widely used organophosphate insecticide that has been shown to disrupt reproductive function in animals. Although the pathogenesis of Dimethoate-induced reproductive toxicity remains to be determined, a reduction in serum testosterone levels is thought to play an important role in the development of Dimethoate-induced infertility. Since Leydig cells play a crucial role in male reproductive function by producing testosterone, the mouse MA-10 Leydig tumor cell line was used to determine if Dimethoate can directly block steroid hormone biosynthesis and to identify the site of steroidogenic inhibition. Dimethoate inhibited steroidogenesis in both a dose- and time-dependent manner without affecting total protein synthesis or protein kinase A activity. While it decreased the activity of the P450 side chain cleavage (P450 scc) enzyme, a reduction in the activity of this enzyme alone could not account for the level of Bu(2)cAMP-inhibited progesterone production. Instead, our results suggest that Dimethoate inhibited steroidogenesis primarily by blocking transcription of the steroidogenic acute regulatory (StAR) gene. This finding is significant since StAR protein mediates the rate-limiting and acutely-regulated step in steroidogenesis, the transfer of cholesterol from the outer to the inner mitochondrial membrane. This study indicates that StAR may be an important target for environmental pollutants which disrupt steroidogenesis and impair reproductive function.

Beating rate of isolated neonatal cardiomyocytes is regulated by the stable microtubule subset.

We investigated the roles of microtubule (MT) dynamics (growth and shrinkage), the stable, nongrowing MT subset, the posttranslationally detyrosinated MT subset, and artificially elevated tubulin levels in the negative regulation of heart cell beating rate. We manipulated the MT populations in isolated, neonatal cardiomyocytes obtained from normal animals in several ways and then measured heart cell beating rate directly. We found that the stabilized population of MTs was sufficient to maintain a normal beating rate, whereas MT dynamics and detyrosination made no observable contribution. Furthermore, by directly and acutely increasing the level of tubulin within otherwise normally beating cells, we found that the increased tubulin (and MT) levels further depressed the beating rate. In conclusion, the stabilized MT subset is sufficient to maintain the normal beating rate in these cells, whereas increasing the MT density depresses it.

Decreased gap-junctional communication associated with segregation of the neuronal phenotype in the RT4 cell-line family.

RT4 is a family of cell lines derived from a rat peripheral neurotumor. The RT4 family consists of a multipotential stem-cell line that spontaneously gives rise to three derivative cell types, one glial and two neuronal. The three derivative cell types are capable of further lineage-specific maturation under appropriate culture conditions. Gap-junctional communication is postulated to be important during nervous-system development by allowing and/or controlling the transmission of both electrical current and signaling molecules, which may affect growth and differentiation. Our characterization of gap-junctional communication in the RT4 cell line family revealed that: (1) the glial-derivative and the stem-cell line were extensively coupled, while the two neuronal derivatives were significantly less coupled, and (2) all of the RT4 cell lines, including the stem-cell line, expressed Cx43 mRNA and protein, and the levels were generally consistent with the observed degree of functional coupling. These observations are consistent with data from in vivo studies and establish the RT4 cell line family as a potentially useful in vitro model system for understanding the role(s) of gap-junctional communication during differentiation in the peripheral nervous system.

Neonatal rat cardiomyocytes possess a large population of stable microtubules that is enriched in post-translationally modified subunits.

Microtubules (MTs) may be involved in several differentiation-specific cardiomyocyte functions, including myofibril organization, hypertrophic cell growth, and the negative regulation of heart cell contraction. The functional characteristics of neonatal rat heart cell MTs, which possess subsets that are enriched with either detyrosinated or acetylated tubulin subunits, were analysed. When compared with their non-myocyte neighbors, cardiomyocyte MTs were found to be more resistant to high concentrations (33 microM) of nocodazole (over 10-30 min), cold treatments (10 min to 8 h), or calcium (50-100 microM for 1-10 min, using detergent-extracted cells). These stable MTs were correlated with the modified MT population. Myocytes treated with 10 microM taxol for 4 h showed elevated levels of modified tubulin but only moderate MT bundling or rearrangement, while after an overnight treatment significant changes in intensity and distribution were noted. In contrast, non-myocytes on the same coverslip showed much greater MT rearrangements, but with only a moderate increase in the immunostaining intensity for modified MTs. Finally, hapten-labeled tubulin that was microinjected into the myocytes showed incorporation rates that were similar to those of the non-myocytes in this study as well as to fibroblast rates determined in other studies, suggesting that the modified MTs in cardiomyocytes may be only moderately stable. Thus, a significant subset of MTs in neonatal cardiomyocytes are post-translationally modified, are resistant to depolymerization by a variety of agents, but are still turning over. These MTs may help define myocyte function during heart development.

Regulation of post-translationally modified microtubule populations during neonatal cardiac development.

Microtubules have been implicated in a number of muscle-specific functions, including sarcomerogenesis and the regulation of heart cell beating rate. Post-translationally modified microtubules (MTs) have been correlated, in other cell types, with MTs of increased stability and possibly distinct functions. This study was begun in order to determine whether neonatal heart development is associated with changes in MT populations, as a prelude to assaying their role in cardiac development and function. Biochemical and morphological studies were performed on heart tissues and cells, over the developmental range from embryonic Day 19 through neonatal Day 20, as well as only fully grown adults. The specific activity of the detyrosinating enzyme, tubulin carboxypeptidase, was high in early neonatal hearts but then decreased progressively to adulthood. Levels of tyrosinated, detyrosinated, and total tubulin varied in a complex manner over the same time period, while levels of acetylated tubulin in detergent-extracted homogenates were low in all age groups. Immunofluorescence analysis of heart sections revealed non-uniform levels of Glu and acetylated tubulin between cells over that period. Cultured, neonatal day 3 myocytes exhibited much more prominent populations of both Glu and acetylated MTs than were present in the co-isolated nonmyocytes, while cell cultures from older animals showed more restricted staining for both MT types. The immunostaining pattern for 7-tubulin, a marker for MT-organizing centers, was diffuse in the cardiomyocytes throughout this period, while the staining in the non-myocytes was much more focused and punctate. These results reveal that individual MT populations are present in developing heart tissue, and may be required for specific functions during myocyte differentiation.

Regulation of cytoplasmic tubulin carboxypeptidase activity in vitro by cations and sulfhydryl-modifying compounds.

alpha-tubulin subunits within microtubules (MTs) can be post-translationally detyrosinated by a tubulin-specific carboxypeptidase (TCP) activity to form biochemically distinct MTs. Attempts to characterize and purify TCP have suffered from the inability to detect low levels of activity and to distinguish TCP from other, competing enzyme activities. We recently developed an assay for TCP [Webster et al. (1992) Biochemistry 31:5849] that uses taxol-stabilized MTs as the substrate. In this study, we exploited the increased sensitivity and specificity of this new assay to explore the effects of various agents that might act to either stimulate or inhibit this enzyme in vitro. We tested a variety of both monovalent and divalent cations for their ability to affect TCP, and tested whether the cations were affecting the enzyme, the substrate, or both. We found that TCP displayed salt-sensitive binding to MTs, characteristic of other, more well characterized MT-associated proteins. While both calcium and magnesium stimulated TCP activity over a narrow concentration range (2-10 mM), they inhibited activity at higher concentrations. Other divalent cations tested, including zinc, copper, and cobalt, inhibited TCP at virtually all concentrations tested, but to different levels (zinc > copper > cobalt). Most of the zinc-induced TCP inhibition was attributed to the interference with the normal binding of TCP to MTs. In addition, we examined the involvement of free sulfhydryl groups (which are important for the activities of many types of enzymes) in TCP activity by the addition of sulfhydryl-modifying compounds during the assay, and found that their addition reduced TCP activity mainly (but not solely) by their action on the extract that contained the TCP. Finally, we tested the ability of DL-benzylsuccinic acid, a potent inhibitor of carboxypeptidase A, to inhibit TCP. While carboxypeptidase A has been found, in other studies, to be inhibited by micromolar concentrations, TCP was affected only at concentrations above 20 mM, adding another proof that carboxypeptidase A and TCP are distinct enzyme activities.

Regulation of cytoplasmic tubulin carboxypeptidase activity during neural and muscle differentiation: characterization using a microtubule-based assay.

A cycle of posttranslational modification of alpha-tubulin has previously been described in higher eukaryotes, in which a C-terminal tyrosine residue is removed and replaced by two complementary cytoplasmic enzymes. The activity of the detyrosinating enzyme, tubulin carboxypeptidase (TCP), and its potential for regulating the level of detyrosinated (Glu) subunits in microtubules (MTs) is of great interest, since TCP catalyzes the primary modification of tubulin and since the level of Glu alpha-tubulin in MTs increases during a variety of differentiative and morphogenetic events. As a first step in examining the role of TCP in cellular morphogenesis, it was necessary to develop an assay for TCP with sufficient sensitivity and specificity to detect TCP activity during these events. Unlike previously described assays for TCP, ours makes use of the affinity TCP exhibits for MTs. NGF-induced neurite outgrowth in PC-12 cells was accompanied by a moderate (approximately 2-fold) increase in TCP activity, while myogenesis of L6 cells resulted in an almost insignificant decrease in activity. Measurements of TCP activity during differentiation were correlated with the level of extract Tyr tubulin, which increased (approximately 37%) during neurite outgrowth and was unchanged during myogenic differentiation. Our results suggest that TCP activity is regulated relative to its substrate, Tyr tubulin, and that changes in MT dynamics, rather than enzymatic activities, are the primary determinants of MT posttranslational modification state during differentiation. In addition, the assay we have devised for TCP and the characterization of TCP during differentiation may allow the future delineation of the mechanism(s) of regulation of TCP and the role this enzyme plays in modulating MT function during differentiation.

Detyrosination of alpha tubulin does not stabilize microtubules in vivo.

The relationship between alpha tubulin detyrosination and microtubule (MT) stability was examined directly in cultured fibroblasts by experimentally converting the predominantly tyrosinated MT array to a detyrosinated (Glu) array and then assaying MT stability. MTs in mouse Swiss 3T3 cells displayed an increase in Glu immunostaining fluorescence approximately 1 h after microinjecting antibodies to the tyrosinating enzyme, tubulin tyrosine ligase. Detyrosination progressed to virtual completion after 12 h and persisted for 30-35 h before tyrosinated subunits within MTs were again detected. The stability of these experimentally detyrosinated MTs was tested by first injecting either biotinylated or Xrhodamine-labeled tubulin and then measuring bulk turnover by hapten-mediated immunocytochemistry or fluorescence recovery after photobleaching, respectively. By both methods, turnover was found to be similarly rapid, possessing a half time of approximately 3 min. As a final test of MT stability, the level of acetylated tubulin staining in antibody-injected cells was compared with that observed in adjacent, uninjected cells and also with the staining observed in cells whose MTs had been stabilized with taxol. Although intense Glu staining was observed in both injected and taxol-treated cells, increased acetylated tubulin staining was observed only in the taxol-stabilized MTs, indicating that the MTs were not stabilized by detyrosination. Together, these results demonstrated clearly that detyrosination does not directly confer stability on MTs. Therefore, the stable MTs observed in these and other cell lines must have arisen by another mechanism, and may have become posttranslationally modified after their stabilization.

Microtubules are acetylated in domains that turn over slowly.

Tubulin is subject to a post-translational acetylation reaction that is thought to be correlated with increased stability of the modified microtubules (MTs). We sought to test directly the stability of acetylated MTs by determining their specific rate of turnover. We used human fibroblasts, which contain a subset of MTs that display terminal and internal domains of acetylation. The turnover of acetylated domains was analysed by microinjecting cells with biotinylated brain tubulin and determining, by triple-label immunofluorescence, the progress of incorporation of biotinylated tubulin into acetylated and non-acetylated domains. Within two minutes after injection, biotinylated domains were contiguous with virtually all observed non-acetylated MT ends but were not contiguous with terminal acetylated domains, demonstrating that the former were growing while the latter were not. Ten minutes after injection, many MTs lacking acetylated domains had incorporated biotinylated subunits uniformly while most MTs containing acetylated domains remained unlabelled, indicating that non-acetylated MTs were turning over while most acetylated domains were not. One hour after injection, virtually all non-acetylated MTs were labelled with biotin whereas approximately half of the acetylated domains contained biotin, demonstrating that acetylated domains turned over much more slowly than the non-acetylated, bulk array. Non-acetylated MT regions flanking acetylated domains also lacked hapten, indicating that acetylation modified discrete regions along stable MTs. Sixteen hours after injection, cells that had not entered mitosis still retained acetylated domains that had not turned over (13% of all acetylated domains), indicating that acetylated domains can be extremely long-lived.(ABSTRACT TRUNCATED AT 250 WORDS)