Highly processive motility is not a general feature of the kinesins

Evidence is presented that the kinesin-related ncd protein is not as processive as kinesin. In low surface density motility experiments, a dimeric ncd fusion protein behaved mechanistically more similar to non-processive myosins than to the highly processive kinesin. First, there was a critical microtubule length for motility; only microtubules longer than this critical length moved in low density ncd surfaces, which suggested that multiple ncd proteins must cooperate to move microtubules in the surface assay. Under similar conditions, native kinesin demonstrated no critical microtubule length, consistent with the behavior of a highly processive motor. Second, addition of methylcellulose to decrease microtubule diffusion decreased the critical microtubule length for motility. Also, the rates of microtubule motility were microtubule length dependent in methylcellulose; short microtubules, that interacted with fewer ncd proteins, moved more slowly than long microtubules that interacted with more ncd proteins. In contrast, short microtubules, that interacted with one or a few kinesin proteins, moved on average slightly faster than long microtubules that interacted with multiple kinesins. We conclude that a degree of processivity as high as that of kinesin, where a single dimer can move over distances on the order of one micrometer, may not be a general mechanistic feature of the kinesin superfamily. Received: 16 September 1997 / Accepted: 4 November 1997     

A TEST OF TWO POSSIBLE MECHANISMS FOR PHOTOTACTIC STEERING IN VOLVOX CARTERI (CHLOROPHYCEAE)

We tested two competing models that could explain how differential flagellar activity leads to phototactic turning in spheroids of Volvox carteri f. weismannia (Powers) Iyengar. In one model, turning results from the flagella of anterior cells in the lighted and shadowed hemispheres beating at different frequencies. In a competing model, turning results from a change in beat direction in these flagella. Both models successfully explain phototactic steering under constant illumination, but they make different predictions when colonies are exposed to abrupt changes in light intensity. If turning is due to control of flagellar beat frequency, both progression and rotation rates will change in the same direction and with similar magnitudes. If spheroid turning is due to a change in flagellar beat direction, a decreased rate of progression will accompany an increased rate of rotation and vice versa. We used video-microscopy to observe the behavior of positively phototactic V. carteri spheroids exposed to 10× step-up and step-down stimuli. After a step-up stimulus, spheroids slow their progression and rotation by equal amounts. No significant changes are reported in these parameters after the reciprocal step-down response. These observations are consistent with the variable flagellar frequency model and inconsistent with the variable flagellar direction model for phototactic turning. Switching the direction of light stimulus by 180° results in reorientation of positively phototactic spheroids. The kinetics of this reorientation did not precisely match the predictions of either model.     

内源性Orexin-A调控大鼠胃运动的中枢及外周机制

目的:探讨内源性Orexin-A(OXA)对大鼠胃运动的中枢和外周作用机制。方法:选取成年Wistar大鼠为研究对象,通过禁食诱导大鼠合成内源性OXA。血浆OXA浓度采用放射免疫法测定。实验前大鼠注射OXA受体拮抗剂SB334867,观察内源性OXA的作用。迷走神经切断术用来观察迷走神经的介导作用。胃排空采用分光光度法测量,消化间期胃运动通过在胃窦部植入一应力传感器测量。Orexin前体(PPO)在胃和下丘脑组织的表达,采用蛋白印迹确定。结果:禁食18 h后,血浆OXA水平和PPO蛋白表达显著增加(P0.05),在禁食36 h组达到最高水平(P0.01)。内源性OXA促进胃排空(P0.05),抑制消化间期胃蠕动(P0.05)。外周注射SB334867均能阻断上述胃动力效应(P0.05),但对PPO表达没有影响。迷走神经切断术不能阻断内源性OXA的介导作用(P0.05)。结论:禁食能诱导内源性OXA的合成,内源性OXA能加速胃排空,同时它又抑制消化间期胃蠕动。     

Thrombospondin‐1 is part of a Slug‐independent motility and metastatic program in cutaneous melanoma,in association with VEGFR‐1 and FGF‐2

Differently from most transformed cells, cutaneous melanoma expresses the pleiotropic factor thrombospondin‐1 (TSP‐1). Herein, we show that TSP‐1 (RNA and protein), undetectable in four cultures of melanocytes and a RGP melanoma, was variously present in 13 cell lines from advanced melanomas or metastases. Moreover, microarray analysis of 55 human lesions showed higher TSP‐1 expression in primary melanomas and metastases than in common and dysplastic nevi. In a functional enrichment analysis, the expression of TSP‐1 correlated with motility‐related genes. Accordingly, TSP‐1 production was associated with melanoma cell motility in vitro and lung colonization potential in vivo. VEGF/VEGFR‐1 and FGF‐2, involved in melanoma progression, regulated TSP‐1 production. These factors were coexpressed with TSP‐1 and correlated negatively with Slug (SNAI2), a cell migration master gene implicated in melanoma metastasis. We conclude that TSP‐1 cooperates with FGF‐2 and VEGF/VEGFR‐1 in determining melanoma invasion and metastasis, as part of a Slug‐independent motility program.     

Reproductive and Developmental Toxicity of Orally Administered Botanical Composition,UP446‐Part III: Effects on Fertility and Early Embryonic Development to Implantation in Sprague Dawley Rats

In recent years, high prevalence of adverse effects associated to the use of traditional medicines during pregnancy is becoming alarming due to the self‐medication of oral supplements by expecting mothers without supervision. Many expectant mothers use alternative and complementary medicines as a supplement to conventional pregnancy management with an inherent belief of considering herbal remedies as harmless. To the contrary, herbal remedies could incur a potential teratogenic risk both to the child bearing mother and the developing fetuses when consumed before or at the time of gestation. Here, we describe the potential adverse effects of orally administered UP446, a standardized bioflavonoid composition from the roots of Scutellaria baicalensis and the heartwoods of Acacia catechu, on fertility and early embryonic development to implantation in Sprague Dawley rats at doses of 250, 500, and 1000 mg/kg. Besides body weight and food consumption, reproductive functions, sperm motility and morphology, estrus cycle, and fertility rate were monitored. There were no statistically significant differences in reproductive function in all UP446 treated groups in both genders. Test substance impacts on reproductive parameters were very minimal. Neither sperm motility nor morphology was affected as a result of oral UP446 administrations in males. There were no treatment‐related effects on estrus cycle stages in females. No significant changes in necropsy or histopathology were observed for all the groups. Therefore, the no observed adverse effect level (NOAEL) of UP446 was considered to be 1000 mg/kg, the highest dose tested, in both genders     

Traction force microscopy in rapidly moving cells reveals separate roles for ROCK and MLCK in the mechanics of retraction

Retraction is a major rate-limiting step in cell motility, particularly in slow moving cell types that form large stable adhesions. Myosin II dependent contractile forces are thought to facilitate detachment by physically pulling up the rear edge. However, retraction can occur in the absence of myosin II activity in cell types that form small labile adhesions. To investigate the role of contractile force generation in retraction, we performed traction force microscopy during the movement of fish epithelial keratocytes. By correlating changes in local traction stress at the rear with the area retracted, we identified four distinct modes of retraction. “Recoil” retractions are preceded by a rise in local traction stress, while rear edge is temporarily stuck, followed by a sharp drop in traction stress upon detachment. This retraction type was most common in cells generating high average traction stress. In “pull” type retractions local traction stress and area retracted increase concomitantly. This was the predominant type of retraction in keratocytes and was observed mostly in cells generating low average traction stress. “Continuous” type retractions occur without any detectable change in traction stress, and are seen in cells generating low average traction stress. In contrast, to many other cell types, “release” type retractions occur in keratocytes following a decrease in local traction stress. Our identification of distinct modes of retraction suggests that contractile forces may play different roles in detachment that are related to rear adhesion strength. To determine how the regulation of contractility via MLCK or Rho kinase contributes to the mechanics of detachment, inhibitors were used to block or augment these pathways. Modulation of MLCK activity led to the most rapid change in local traction stress suggesting its importance in regulating attachment strength. Surprisingly, Rho kinase was not required for detachment, but was essential for localizing retraction to the rear. We suggest that in keratocytes MLCK and Rho kinase play distinct, complementary roles in the respective temporal and spatial control of rear detachment that is essential for maintaining rapid motility.     

How tropomyosin regulates lamellipodial actin‐based motility: a combined biochemical and reconstituted motility approach

At the leading edge of migrating cells, protrusive forces are developed by the assembly of actin filaments organised in a lamellipodial dendritic array at the front and a more distal lamellar linear array. Whether these two arrays are distinct or functionally linked and how they contribute to cell migration is an open issue. Tropomyosin severely inhibits lamellipodium formation and facilitates the lamellar array while enhancing migration, by a mechanism that is not understood. Here we show that the complex in vivo effects of tropomyosin are recapitulated in the reconstituted propulsion of neural Wiskott–Aldrich syndrome protein (N‐WASP)‐functionalised beads, which is based on the sole formation of a dendritic array of actin‐related protein (Arp)2/3‐branched filaments. Actin‐depolymerising factor (ADF) and tropomyosin control the length of the actin tail. By competing with Arp2/3 during filament branching, tropomyosin displays opposite effects on propulsion depending on the surface density of N‐WASP. Tropomyosin binding to the dendritic array is facilitated following filament debranching, causing its enrichment at the rear of the actin tail, like in vivo. These results unveil the mechanism by which tropomyosin generates two morphologically and dynamically segregated actin networks from a single one.     

Snake venoms as a source of compounds modulating sperm physiology: Secreted phospholipases A2 from Oxyuranus scutellatus scutellatus impact sperm motility,acrosome reaction and in vitro fertilization in mice

The goal of this study was to identify new compounds from venoms able to modulate sperm physiology and more particularly sperm motility. For this purpose, we screened the effects of 16 snake venoms cleared of molecules higher than 15 kDa on sperm motility. Venoms rich in neurotoxins like those from Oxyuranus scutellatus scutellatus or Daboia russelii, were highly potent inhibitors of sperm motility. In contrast, venoms rich in myotoxins like those from Echis carinatus, Bothrops alternatus and Macrovipera lebetina, were inactive. From the main pharmacologically-active fraction of the Taipan snake O. scutellatus s., a proteomic approach allowed us to identify 16 different proteins, among which OS1 and OS2, two secreted phospholipases A2 (sPLA₂). Purified OS1 and OS2 mimicked the inhibitory effect on sperm motility and were likely responsible for the inhibitory effect of the active fraction. OS1 and OS2 triggered sperm acrosome reaction and induced lipid rearrangements of the plasma membrane. The catalytic activity of OS2 was required to modulate sperm physiology since catalytically inactive mutants had no effect. Finally, sperm treated with OS2 were less competent than control sperm to initiate in vitro normal embryo development. This is the first report characterizing sPLA₂ toxins that modulate in vitro sperm physiology.     

The locust foraging gene

Our knowledge of how genes act on the nervous system in response to the environment to generate behavioral plasticity is limited. A number of recent advancements in this area concern food‐related behaviors and a specific gene family called foraging (for), which encodes a cGMP‐dependent protein kinase (PKG). The desert locust (Schistocerca gregaria) is notorious for its destructive feeding and long‐term migratory behavior. Locust phase polyphenism is an extreme example of environmentally induced behavioral plasticity. In response to changes in population density, locusts dramatically alter their behavior, from solitary and relatively sedentary behavior to active aggregation and swarming. Very little is known about the molecular and genetic basis of this striking behavioral phenomenon. Here we initiated studies into the locust for gene by identifying, cloning, and studying expression of the gene in the locust brain. We determined the phylogenetic relationships between the locust PKG and other known PKG proteins in insects. FOR expression was found to be confined to neurons of the anterior midline of the brain, the pars intercerebralis. Our results suggest that differences in PKG enzyme activity are correlated to well‐established phase‐related behavioral differences. These results lay the groundwork for functional studies of the locust for gene and its possible relations to locust phase polyphenism. © 2010 Wiley Periodicals, Inc.