Quantitation and distribution of beta-tubulin in human cardiac myocytes

Increasing evidence suggests that derangements of cytoskeletal proteins contribute to alterations in intracellular signaling, myocyte function, and the coupling of myocytes to the extracellular matrix during cardiac hypertrophy and failure. Data from animal studies have shown an increased density of beta-tubulin protein in the right or left ventricle subjected to pressure overload, and have demonstrated that interfering with excess polymerization of beta-tubulin improves contractility. We tested the hypothesis that beta-tubulin is increased in human left ventricular hypertrophy and end-stage heart failure. Confocal microscopy of fluorescently labeled beta-tubulin protein revealed an increased density of the beta-tubulin network in cardiomyocytes from both hypertrophied and failing human hearts as compared to cells from nonfailing hearts. Western blot analysis on total heart homogenate showed no change in beta-tubulin when data were normalized to either actin or calsequestrin, although there was a significant increase in failing human hearts when data were normalized only for a constant amount of protein per heart. The mRNA for beta-tubulin was not changed in hypertrophied hearts, but was significantly decreased in failing human hearts. Thus, similar to animal models, we have shown that the density of the microtubular network within the cardiomyocyte is increased in end-stage failing human hearts. We have also shown for the first time that beta-tubulin density is increased in cells from hypertrophied human hearts. Although the functional implications of this finding in the human heart remain to be explored, data from animal studies suggest that increased beta-tubulin protein contributes to cardiac dysfunction.     

Carbon nanotube-based substrates for modulation of human pluripotent stem cell fate

We investigated the biological response of human pluripotent stem cells (hPSCs) cultured on a carbon nanotube (CNT) array-based substrate with the long term goal to direct hPSC germ layer specification for a wide variety of tissue engineering applications. CNT arrays were fabricated using a chemical vapor deposition system allowing for control over surface roughness and mechanical stiffness. Our results demonstrated that hPSCs readily attach to hydrophilized and extracellular matrix coated CNT arrays. hPSCs cultured as colonies in conditions supporting self-renewal demonstrated the morphology and marker expression of undifferentiated hPSCs. Conditions inducing spontaneous differentiation lead to hPSC commitment to all three embryonic germ layers as assessed by immunostaining and RT-PCR analysis. Strikingly, the physical characteristics of CNT arrays favored mesodermal specification of hPSCs. This is contradictory to the behavior of hPSCs on traditional tissue culture plastic which promotes the development of ectoderm. Altogether, these results demonstrate the potential of CNT arrays to be used in the generation of new platforms that allow for precise control of hPSC differentiation by tuning the characteristics of their physical microenvironment.     

Palytoxins and cytoskeleton: An overview

Cytoskeleton is a dynamic structure essential for a wide variety of normal cellular processes, including the maintenance of cell shape and morphology, volume regulation, membrane dynamics and signal transduction. Cytoskeleton is organized into microtubules, actin meshwork and intermediate filaments. Actin has been identified as a major target for destruction during apoptosis and is also important under pathological conditions such as cancers.Several natural compounds actively modulate actin organization by specific signaling cascades being useful tools to study cytoskeleton dynamics.Palytoxin is a large bioactive compound, first isolated from zoanthids, with a complex structure and different analogs such as ostreocin-D or ovatoxin-a. This toxin has been identified as a potent tumor promoter and cytotoxic molecule, which leads to actin filament distortion and triggers cell death or apoptosis. In this review we report the findings on the involvement of palytoxin and analogues modulating the actin cytoskeleton within different cellular models.     

Y-27632, a Rho-associated protein kinase inhibitor,attenuates neuronal cell death after transient retinal ischemia

Purpose Transient retinal ischemia induces the death of retinal neuronal cells. Postischemic damage is associated with the infiltration of leukocytes into the neural tissue through vascular endothelia. The current study aimed to investigate whether this damage was attenuated by the inhibition of Rho/ROCK (Rho kinases) signaling, recently shown to play a critical role in the transendothelial migration of leukocytes. Methods Y-27632, a selective inhibitor of ROCK, was injected intravitreally into rat eyes with transient retinal ischemia. Cell loss of the ganglion cell layer (GCL) and thinning of the inner plexiform layer (IPL) with and without the administration of Y-27632 were evaluated by histological anaysis, TUNEL assay and retrograde labeling of retinal ganglion cells (RGCs). To examine the attenuation of leukocyte infiltration in postischemic retinas with the administration of Y-27632, silver nitrate staining and immunohistochemistry using an anti-LCA antibody were performed. Results Cell loss of the GCL and thinning of the IPL were significantly attenuated when 100 nmol Y-27632 was administered within three hours of the induction of ischemia. TUNEL assay and retrograde labeling of RGCs showed a decreased number of apoptotic cells and an increased number of RGCs in Y-27632-injected retinas. Moreover, silver nitrate staining and immunohistochemical analysis using an anti-LCA antibody showed that Y-27632 injection dramatically inhibited leukocyte infiltration and endothelial disarrangement. Conclusions Our data suggest that inhibition of Rho/ROCK signaling offers neuroprotective therapy against postischemic neural damage, by regulating leukocyte infiltration in the neural tissue.     

膜-细胞骨架连接分子Ezrin与肿瘤转移关系的研究进展

Ezrin是埃兹蛋白/根蛋白/膜突蛋白(ERM)家族成员之一,是一种膜细胞骨架连接蛋白。近年来的研究发现,Ezrin在多种恶性肿瘤细胞中异常表达,提示其在肿瘤的浸润、转移机制中发挥着重要作用。Ezrin通过改变肿瘤细胞运动、调节细胞间黏附、参与肿瘤细胞内信号转导、抑制细胞凋亡和调理吞噬细胞的吞噬功能等方面,影响恶性肿瘤的转移。     

Effect of hyperthermia on the transport of mRNA encoding the extracellular matrix glycoprotein SC1 into Bergmann glial cell processes

SC1 is an extracellular matrix glycoprotein that is related to the multifunctional protein SPARC. These matricellular members play regulatory roles in modulating cellular interactions. SC1 expression is enriched in the central nervous system during embryonic and postnatal development as well as in the adult brain. In the rat cerebellum, SC1 is expressed at high levels in Bergmann glial cells and their radial fibers which project into the synaptic-rich molecular layer. At specific stages of development and in the adult, SC1 mRNA is selectively transported into cellular processes of these cells. In the present study, we have examined the effect of whole-body hyperthermia on the transport of SC1 mRNA in Bergmann glial cells of the rat cerebellum. Our results show that SC1 mRNA transport is diminished at 10 and 15 h post-hyperthermia, but returns to control levels by 24 h after heat shock. One of the characteristics of a heat shock on cells grown in tissue culture is a collapse of the cytoskeletal network. Intact components of the cytoskeleton are necessary for the transport of mRNA into peripheral processes of cells. However, in vivo hyperthermia does not appear to affect the morphology of the intermediate filament proteins GFAP, vimentin, or the beta-tubulin component of microtubules in Bergmann glial cell processes. During the hyperthermic time course, levels of vimentin protein increase, which is reflected by immunoreactivity of activated astrocytes and microvasculature in cerebellar white matter.     

Induction of peripherin expression in subsets of brain neurons after lesion injury or cerebral ischemia

Peripherin is a type III intermediate filament predominantly expressed in neurons having direct axonal projections toward peripheral structures. Here, we report that brain injuries can trigger expression of peripherin and the formation of peripherin accumulations in neurons that are normally silent for this gene. Stab lesions made with nitrocellulose implants induced within 4 days the formation of peripherin accumulations, devoid of neurofilament proteins, in thalamic neurites at the site of the lesion. The local administration of interleukin-6 or leukemia inhibitory factor at the site of the stab lesion extended the expression pattern of peripherin to other neuronal subsets in areas of the cortex and/or of the hippocampus adjacent to injury. We also show that transient focal ischemia in mice, a model of stroke, can trigger within 72 h the formation of neuronal peripherin accumulations in neurons of the cortex, thalamus and hippocampus. This new type of potentially noxious intermediate filament protein accumulations, composed of peripherin, may be of relevance to many brain degenerative disorders with occurrence of proinflammatory cytokines.     

组织工程化神经研究进展

近年来,组织工程化周围神经研制工作取得了很大的进展。自体神经、同种异体神经和合成材料均可作为桥接神经缺损的神经导管,以化学萃取的同种异体神经及可降解的生物材料导管较为理想。同时,经培养和纯化后的雪旺细胞具有分泌多种神经营养因子活性,是提高修复神经损伤效果的关键。     

The effect of brown spider venom on endothelial cell morphology and adhesive structures

Spiders of the Loxosceles genus have been responsible for severe clinical cases of envenomation worldwide. Accidents involving brown spiders can cause dermonecrotic injury, hemorrhage, hemolysis, platelet aggregation and renal failure. Histological findings of animals treated by venom have shown subendothelial blebs, vacuoles and endothelial cell membrane degeneration of blood vessel walls, as well as fibrin and thrombus formation. The mechanisms by which the venom causes these disorders are poorly understood. In this work, with an endothelial cell line derived from rabbit aorta, we were able to demonstrate that venom binds to the cell surface and the extracellular matrix. Moreover, we observed that the venom also induced morphological alterations, such as cell retraction, homophilic disadhesion and an increasing in filopodia projections. We also demonstrated that toxins present in the venom disorganized focal adhesion points and actin microfilaments of endothelial cells. Nevertheless, endothelial cell viability showed no alterations compared to controls. Additionally, venom treatment changed the fibronectin matrix profile synthesized by these cells as well as cell adhesion to fibronectin. These results suggest that the deleterious effects of venom on blood vessel walls could be a consequence of the direct effect on the endothelial cell surface and adhesive structures involved in blood vessel stability. These effects indirectly lead to leukocyte and platelet activation, disseminated intravascular coagulation and an increase in vessel permeability.     

Neurofilaments assume a less random architecture at nodes and in other regions of axonal compression

Neurofilament distributions were mathematically characterized in four chicken somatic motor axons at each of four histologically distinct regions: compact myelinated regions, compact myelinated regions associated with Schwann cell nuclei, Schmidt-Lanterman clefts, and nodes of Ranvier. Compact myelinated regions had the largest cross-sectional areas, the lowest neurofilament densities, and the most random neurofilament organizations — nodes of Ranvier had the smallest cross-sectional areas, the highest neurofilament densities, and the most ordered architectures. In these myelinated axons, the closest natural neurofilament spacing was 25 nm. Mathematical analyses of serial sections suggested that neurofilament interactions are sufficiently weak and transient to permit a full range of variation from random to ordered cytoskeletal architectures as the neurofilaments move longitudinally through the few micron span of the paranodal-nodal region of a single axon.