Probing FtsZ and tubulin with C8-substituted GTP analogs reveals differences in their nucleotide binding sites

The cytoskeletal proteins, FtsZ and tubulin, play a pivotal role in prokaryotic cell division and eukaryotic chromosome segregation, respectively. Selective inhibitors of the GTP-dependent polymerization of FtsZ could constitute a new class of antibiotics, while several inhibitors of tubulin are widely used in antiproliferative therapy. In this work, we set out to identify selective inhibitors of FtsZ based on the structure of its natural ligand, GTP. We found that GTP analogs with small hydrophobic substituents at C8 of the nucleobase efficiently inhibit FtsZ polymerization, whereas they have an opposite effect on the polymerization of tubulin. The inhibitory activity of the GTP analogs on FtsZ polymerization allowed us to crystallize FtsZ in complex with C8-morpholino-GTP, revealing the binding mode of a GTP derivative containing a nonmodified triphosphate chain.     

Application of two-dimensional gel electrophoresis in the study of cytoskeletal protein regulation during growth activation and differentiation

Two-dimensional gel electrophoresis was used to study the regulation of cytoskeletal protein synthesis during growth activation and development of the differentiated phenotype. We demonstrated a correlation between the state of organization and the expression of the respective cytoskeletal protein by showing that depolymerization of microtubules leads to a rapid decrease in new tubulin synthesis. We found that the synthesis of vimentin in both fibroblasts and epithelial cells correlates with extensive cell spreading on the substrate, while cytokeratin synthesis is maximal when cell to cell contacts are abundant. The analysis of cytoskeletal elements, involved directly in the formation of cell contacts, revealed that the level of vinculin synthesis is dependent on the extent of adherent type of cell contacts formed. Moreover, we found that the transient disappearance of vinculin from adhesion plaques of quiescent fibroblasts in response to serum factors was followed by an induction of vinculin mRNA and protein synthesis. The morphological changes associated with establishment of the differentiated phenotype were also found to include changes in the expression of the cytoskeletal-extracellular matrix complex. This was demonstrated in several differentiating systems: in 3T3 preadipocytes which change their shape from a fibroblastic to a spherical shape when stimulated to differentiate with adipogenic medium, we observed a decrease in mRNA levels and in the synthesis of fibronectin, beta-integrin, and the microfilament proteins, vinculin, alpha-actinin, tropomyosin and actin. The culturing of these cells on a certain extracellular matrix prevented the morphological changes occurring in the presence of adipogenic medium and blocked the shifts in cytoskeletal- and differentiation-related gene expression. Similar changes in the organization and expression of cytoskeletal proteins were identified during maturation of primary ovarian granulosa cell cultures, stimulated with gonadotropic hormones to form highly steroidogenic cells. The cell rounding and aggregation occurring during this process were associated with a decreased synthesis of vinculin, alpha-actinin, actin and the nonmuscle tropomyosins. The physiological relevance of these changes was suggested by the observation that the level of tropomyosin mRNA was lower in follicles of animals at late stages of granulosa cell maturation when compared to earlier stages. The expression of tissue-specific and cytoskeletal proteins was also determined in primary cultures of liver hepatocytes, maintained under conditions either favorable for growth or for expression of liver-specific functions. When DNA synthesis was elevated, cytoskeletal protein synthesis was high and that of liver-specific proteins was low.(ABSTRACT TRUNCATED AT 400 WORDS)     

Betulonic Acid,as One of the Active Components of the Celastrus orbiculatus Extract,Inhibits the Invasion and Metastasis of Gastric Cancer Cells by Mediating Cytoskeleton Rearrangement In Vitro

Gastric cancer is a type of malignant tumor that seriously threatens human life and health. Invasion and metastasis present difficulties in the treatment of gastric cancer, and the remodeling of the tumor cytoskeleton plays an important role in mediating the ability of tumor cells to achieve invasion and metastasis. Previous experimental results suggest that Celastrus orbiculatus extract can regulate cytoskeletal remodeling in gastric cancer, but the active component has not been determined. Betulonic acid, as an effective component of COE, inhibits the invasion and metastasis of gastric cancer cells by regulating cytoskeletal remodeling in vitro; its specific mechanisms have been studied here. After betulonic acid was dissolved, it was diluted to various working concentrations in RPMI-1640 medium and added to AGS, HGC-27 and GES-1 cell lines. Cell viability was assessed by CCK-8 and colony formation assays. Cytoskeleton staining was used to detect changes in cytoskeleton morphology. Functional assays including wound healing assays and transwell assays were used to detect the invasion and migration of cells. The effect of betulonic acid on cell invasion and migration was clearly and precisely observed by high-content imaging technology. Western blotting was used to detect the regulation of matrix metalloproteinase-related proteins and epithelial–mesenchymal transformation-related proteins. We found that betulonic acid inhibited the migration and invasion of gastric cancer cells. Therefore, betulonic acid inhibits the invasion and metastasis of gastric cancer cells by mediating cytoskeletal remodeling and regulating epithelial mesenchymal transformation.     

Quantifying blood platelet morphological changes by dissipation factor monitoring in multilayer shells

The ability of electrostatically driven layer-by-layer (LbL) assembly to adapt to the morphological features of a template was explored. Subtle cytoskeletal changes in blood platelets became traceable through energy dissipation monitoring in multilayered shells using microgravimetric measurements. This LbL coating was sequentially deposited on protein-modified chips onto which platelets were adhered. In addition to consequently improving the signal sensitivity, the LbL shell acted in synergy with the cell, allowing the determination and quantification of cytoskeletal changes induced by the specific cell adhesion to the protein-modified chip surface used with a quartz crystal microbalance with dissipation. The difference in cell morphology, as a result of the optimization of specific interactions between the protein layer and cell membrane integrins induced viscoelastic changes in the polyelectrolyte shell, thereby providing quantitative data on platelet conformational changes upon their adhesion to protein-modified chip surface.     

Computer-aided molecular design of (E)-N-Aryl-2-ethene-sulfonamide analogues as microtubule targeted agents in prostate cancer

Microtubules are tube-shaped, filamentous and cytoskeletal proteins that are essential in all eukaryotic cells. Microtubule is an attractive and promising target for anticancer agents. In this study, three-dimensional quantitative structure activity relationships (3D-QSAR) including comparative molecular field analysis, CoMFA, and comparative molecular similarity indices analysis, CoMSIA, were performed on a set of 45 (E)-N-Aryl-2-ethene-sulfonamide analogues as microtubule-targeted anti-prostate cancer agents. Automated grid potential analysis, AutoGPA module in Molecular Operating Environment 2009.10 (MOE) as a new 3D-QSAR approach with the pharmacophore-based alignment was carried out on the same dataset. AutoGPA-based 3D-QSAR model yielded better prediction parameters than CoMFA and CoMSIA. Based on the contour maps generated from the models, some key features were identified in (E)-N-Aryl-2-arylethene-sulfonamide analogues that were responsible for the anti-cancer activity. Virtual screening was performed based on pharmacophore modeling and molecular docking to identify the new inhibitors from ZINC database. Seven top ranked compounds were found based on Gold score fitness function. In silico ADMET studies were performed on compounds retrieved from virtual screening in compliance with the standard ranges.     

Cross-linking of CD4 induces cytoskeletal association of CD4 and p56lck

A membrane glycoprotein CD4 functions as a co-receptor of a T lymphocyte. The co-receptor function has been attributed to a protein tyrosine kinase, p56lck, which is activated upon CD4 binding to MHC molecule. In this study, we present evidences that one of the pathways through which CD4 transmits its signal is cytoskeleton association of p56lck tyrosine kinase as well as CD4 itself. Cytoskeletal association of both proteins is inhibited by a tyrosine kinase inhibitor, genistein, indicating that tyrosine protein kinase activation is important for cytoskeletal association of CD4 and p56lck. Cytoskeletal association of these proteins by CD4 cross-linking is not affected by inhibitors of protein kinase C nor PI3-kinase. Taken together, these results suggest that CD4 cross-linking activates a tyrosine kinase which then induces the simultaneous association of CD4 and p56lck with cytoskeleton.     

Single-cell electrical lysis of erythrocytes detects deficiencies in the cytoskeletal protein network

The network of erythrocyte cytoskeletal proteins significantly influences erythrocyte physical and biological properties. Here we show that the kinetics of erythrocyte lysis during exposure to an electric field is sensitively correlated with defects in the cytoskeletal network. Histograms compiled from single-cell electrical lysis data show characteristics of erythrocyte populations that are deficient in a specific cytoskeletal protein, revealing the presence of cell subpopulations.     

The use of neural networks and texture analysis for rapid objective selection of regions of interest in cytoskeletal images

Understanding cytoskeletal dynamics in living tissue is prerequisite to understanding mechanisms of injury, mechanotransduction, and mechanical signaling. Real-time visualization is now possible using transfection with plasmids that encode fluorescent cytoskeletal proteins. Using this approach with the muscle-specific intermediate filament protein desmin, we found that a green fluorescent protein-desmin chimeric protein was unevenly distributed throughout the muscle fiber, resulting in some image areas that were saturated as well as others that lacked any signal. Our goal was to analyze the muscle fiber cytoskeletal network quantitatively in an unbiased fashion. To objectively select areas of the muscle fiber that are suitable for analysis, we devised a method that provides objective classification of regions of images of striated cytoskeletal structures into "usable" and "unusable" categories. This method consists of a combination of spatial analysis of the image using Fourier methods along with a boosted neural network that "decides" on the quality of the image based on previous training. We trained the neural network using the expert opinion of three scientists familiar with these types of images. We found that this method was over 300 times faster than manual classification and that it permitted objective and accurate classification of image regions.     

Unconventional specimen preparation techniques using high resolution low voltage field emission scanning electron microscopy to study cell motility, host cell invasion, and internal cell structures in Toxoplasma gondii.

Apicomplexan parasites employ complex and unconventional mechanisms for cell locomotion, host cell invasion, and cell division that are only poorly understood. While immunofluorescence and conventional transmission electron microscopy have been used to answer questions about the localization of some cytoskeletal proteins and cell organelles, many questions remain unanswered, partly because new methods are needed to study the complex interactions of cytoskeletal proteins and organelles that play a role in cell locomotion, host cell invasion, and cell division. The choice of fixation and preparation methods has proven critical for the analysis of cytoskeletal proteins because of the rapid turnover of actin filaments and the dense spatial organization of the cytoskeleton and its association with the complex membrane system. Here we introduce new methods to study structural aspects of cytoskeletal motility, host cell invasion, and cell division of Toxoplasma gondii, a most suitable laboratory model that is representative of apicomplexan parasites. The novel approach in our experiments is the use of high resolution low voltage field emission scanning electron microscopy (LVFESEM) combined with two new specimen preparation techniques. The first method uses LVFESEM after membrane extraction and stabilization of the cytoskeleton. This method allows viewing of actin filaments which had not been possible with any other method available so far. The second approach of imaging the parasite's ultrastructure and interactions with host cells uses semithick sections (200 nm) that are resin de-embedded (Ris and Malecki, 1993) and imaged with LVFESEM. This method allows analysis of structural detail in the parasite before and after host cell invasion and interactions with the membrane of the parasitophorous vacuole as well as parasite cell division.     

Discovery of Potent, Selective and Reversible Caspase-3 Inhibitors

Recent studies towards understanding the molecular mechanisms of apoptosis have revealed the importance of a group of cysteinyl aspartate specific proteases, the caspases, in the programmed cell death process (Hengartner, M.O. Nature 2000, 407, 770). Caspase-3, in particular, has been characterized as the dominant effector caspase involved in the proteolytic cleavage of a variety of protein substrates including cytoskeletal proteins, kinases and DNA repair enzymes during apoptosis (Nicholson…     

Cytoskeleton mediated effective elastic properties of model red blood cell membranes

The plasma membrane of human red blood cells consists of a lipid bilayer attached to a regular network of underlying cytoskeletal polymers. We model this system at a dynamic coarse-grained level, treating the bilayer as an elastic sheet and the cytoskeletal network as a series of phantom entropic springs. In contrast to prior simulation efforts, we explicitly account for dynamics of the cytoskeletal network, both via motion of the protein anchors that attach the cytoskeleton to the bilayer and through breaking and reconnection of individual cytoskeletal filaments. Simulation results are explained in the context of a simple mean field percolation model and comparison is made to experimental measurements of red blood cell fluctuation amplitudes.     

Differential detergent fractionation of isolated hepatocytes: Biochemical,immunochemical and two-dimensional gel electrophoresis characterization of cytoskeletal and noncytoskeletal compartments

Two-dimensional (2-D) gel electrophoresis is often used in toxicologic and metabolic studies to assess treatment- or stage-specific changes in protein synthesis, degradation or posttranslational modification. When combined with cell fractionation studies the detectability of low abundance proteins is enhanced, and changes in subcellular distribution of proteins can also be monitored. Detergent fractionation is a simpler alternative to differential pelleting, which partitions cellular constituents into functionally distinct populations while preserving cytoskeletal integrity. We defined and characterized a differential detergent fractionation (DDF) protocol to enable protein dynamics in cytoskeletal and noncytoskeletal compartments of isolated hepatocytes to be monitored simultaneously. Rat hepatocytes were maintained in suspension culture and fractionated by sequential extraction with detergentcontaining buffers (digitonin/EDTA, Triton/EDTA, Tween/deoxycholate). DDF reproducibly yielded four electrophoretically distinct fractions enriched in cytosolic, membrane-organelle, nuclear membrane and cytoskeletal-matrix markers, respectively. Immunoblotting with over 20 different antibodies corroborated the selectivity of fractionation and was used to characterize the distribution profiles of cytoskeletal (actin, tubulins, cytokeratins, vinculin, myosin, desmoplakins, fodrin, nuclear lamins) and noncytoskeletal proteins (heat-shock 70 proteins, glutathione-S-transferase, calpains, carbamoyl phosphate synthetase, etc.), as well as to identify spots in 2-D gels. Detergent buffers were compatible with equilibrium or nonequilibrium 2-D gel electrophoretic analysis. Extensive 2-D maps of acidic and basic proteins in each fraction were generated along with a tabular listing of M_r and pI. Thus, DDF reproducibly partitions hepatocytic proteins into functionally distinct cytoskeletal and noncytoskeletal compartments that are readily analyzed by 2-D gel electrophoresis. DDF is simple, applicable to use with other cell types or culture systems and is especially useful when biomaterial is limited (i.e., clinical studies).     

Heterocyclic inflammation inhibitors

Non steroidal anti-inflammatory drugs are the most widely used medicines for relief of pain. These drugs have some side effects, particularly toxicity in the gastrointestinal tract and kidneys. Various approaches have been used for obtaining safer anti-inflammatory drugs. In this review we have summarized the recent developments in the following areas; (i) mode of action of NSAIDs (ii) Role of COX-1 & COX-2 in inflammation, (iii) Different approaches used to improve gastric tolerance i.e. chemical manipulation, formulation & co-administration, development of non specific (COX-1 & COX-2 inhibitors) and specific (COX-2 inhibitors) inflammation inhibitors, and development of inflammation inhibitors having a mode of action other than COX-1 & COX-2 inhibition. We have also focused on the safety of COX-2 inhibitors and the synthesis of heterocyclic compounds and their role as inflammation inhibitors.     

Interaction of liposomes with cultured cells: possible involvement of lipid peroxidation in cell growth inhibition

Systematic analyses of the interaction between liposomes and cells were examined. Liposomes were found to affect the growth of mouse NIH 3T3 cells depending upon their size, net charge, and cholesterol content. Among the charged compounds, stearylamine was the most inhibitory and showed complete inhibition of cell growth at 100 microM. The cholesterol-rich and small unilamellar vesicles were more suppressive compared to the cholesterol-poor and multilamellar ones, respectively. The binding assay of liposomes to the cells showed a positive correlation between liposome binding and the extent of growth inhibition. Suppression of liposome uptake by inhibitors of the cytoskeletal system and energy metabolism were suggestive of an endocytotic mechanism for the cellular uptake of liposomes. The growth inhibitory effect seemed secondary to the intracellular uptake of liposomes, and peroxidation of incorporated lipids would lead to cellular damage. Therefore, it is highly recommended that potential growth inhibitory effects associated with the particular composition and other properties of liposomes should be carefully assessed in any human studies, especially for long-term use.     

Site-directed photochemical disruption of the actin cytoskeleton by actin-binding Rose Bengal-conjugates

The in situ light-induced, non-enzymatic digestion of cytoskeletal actin by a xanthene dye conjugated to heavy meromyosin, anti-actin antibodies and/or anti-myosin antibodies is reported. The dye Rose Bengal was conjugated to either anti-actin antibodies, anti-myosin antibodies or heavy meromyosin. Under our experimental conditions, visible light induced the non-enzymatic breakdown of cytoskeletal actin when mammalian tissue culture cells were probed either with Rose Bengal-conjugated anti-actin and/or anti-myosin antibodies. Similar results were obtained when tissue culture cells were probed with Rose Bengal-conjugated heavy meromyosin before irradiation with visible light. The in situ photochemical reaction depended on the presence of actin-binding Rose Bengal-conjugates.     

Diffusion dynamics of motor-driven transport: gradient production and self-organization of surfaces

The interaction between cytoskeletal filaments (e.g., actin filaments) and molecular motors (e.g., myosin) is the basis for many aspects of cell motility and organization of the cell interior. In the in vitro motility assay (IVMA), cytoskeletal filaments are observed while being propelled by molecular motors adsorbed to artificial surfaces (e.g., in studies of motor function). Here we integrate ideas that cytoskeletal filaments may be used as nanoscale templates in nanopatterning with a novel approach for the production of surface gradients of biomolecules and nanoscale topographical features. The production of such gradients is challenging but of increasing interest (e.g., in cell biology). First, we show that myosin-induced actin filament sliding in the IVMA can be approximately described as persistent random motion with a diffusion coefficient (D) given by a relationship analogous to the Einstein equation (D = kT/gamma). In this relationship, the thermal energy (kT) and the drag coefficient (gamma) are substituted by a parameter related to the free-energy transduction by actomyosin and the actomyosin dissociation rate constant, respectively. We then demonstrate how the persistent random motion of actin filaments can be exploited in conceptually novel methods for the production of actin filament density gradients of predictable shapes. Because of regularly spaced binding sites (e.g., lysines and cysteines) the actin filaments act as suitable nanoscale scaffolds for other biomolecules (tested for fibronectin) or nanoparticles. This forms the basis for secondary chemical and topographical gradients with implications for cell biological studies and biosensing.     

Deforming biological membranes: how the cytoskeleton affects a polymerizing fiber

We give a theoretical treatment of the force exerted by a fluctuating membrane on a polymer rod tip, taking into account the effects of an underlying biological cytoskeleton by way of a simple harmonic dependence on displacement. We also consider theoretically and experimentally the dynamics of a growing fiber tip under the influence of such a fluctuation-induced membrane force, including the effects of an underlying cytoskeletal network. We compare our model with new experimental data for the growth of hemoglobin fibers within red blood cells, revealing a good agreement. We are also able to estimate the force and membrane/cytoskeletal displacement required to stall growth of, or buckle, a growing fiber. We discuss the significance of our results in a biological context, including how the properties of the membrane and cytoskeleton relate to the thermodynamics of rod polymerization.     

Design and Synthesis of 5-Lipoxygenase Inhibitors

Based on the substrate specificity for 5-lipoxygenase and the known stereochemical course of the reaction, a hypothetical model of the enzyme active site was developed and used to design 2 types of selective inhibitors of 5-lipoxygenase. Both inhibitor types used aromatic rings in place of ( Z )-olefins of the substrate and were designed to mimic the nonpolar end of arachidonic acid. One inhibitor type used a carboxylic-acid interaction with the O-binding centre of the enzyme in analogy with known cyclooxygenase inhibitors, whereas a second type employed a hydroxylamine function to interact with a presumed tyrosine or cysteinyl radical predicted to be in the enzyme active site. Selective 5-lipoxygenase inhibitors were 7-(hexyloxy) naphthalene-2-acetic acid ( 1 ) and N -methyl;- N (7-propoxynaphthalene-2-ethyl)hydroxylamine ( 2 ). Structure-activity relationships for both types of inhibitors are discussed.     

细胞在单壁碳纳米管无纺膜支架上的生长行为

以具有纳米拓扑结构特征的单壁碳纳米管无纺膜材料为支架, 选择在促进组织修复和再生中起重要作用的成纤维细胞株作为实验细胞, 研究了该材料对细胞生长行为的影响. 通过X射线光电子能谱分析, 表征其在细胞培养液中浸泡后的表面化学组成; 通过细胞粘附、增殖实验以及细胞骨架发育观察, 探讨了材料的微观纳米拓扑结构对细胞的作用, 以及与碳纤维、聚氨酯浇铸膜和空白培养板材料对细胞作用的差异和可能的机理; 并采用双层细胞培养装置, 研究了该材料通过细胞通讯途径对在其它材料上生长的细胞增殖的影响. 实验结果表明, 单壁碳纳米管无纺膜材料为细胞提供了十分接近天然细胞外基质的人造微环境, 具有显著促进细胞粘附和长时间增殖的功能, 而且生长在该支架上的细胞可能通过旁分泌方式将某些化学介质分泌到细胞外液中, 经局部扩散作用于在其它材料上生长的细胞, 促进它们的增殖.