Rearrangement Of Microfilaments Research Articles

F-actin microfilaments affect the LIPUS-promoted osteogenic differentiation of BMSCs through TRPM7.

The differentiation of bone marrow mesenchymal stem cells (BMSCs) toward osteogenesis can be induced by low-intensity pulsed ultrasound (LIPUS). However, themolecular mechanisms responsible for LIPUS stimulation areunclear. The possible molecular mechanisms by which LIPUS promotes osteogenic differentiation of BMSCs were investigated in this study. The quantification of alkaline phosphatase (ALP) activity, Alizarin Red S staining, ALP staining, and the establishment of a calvarial defect model were used to evaluate osteogenic effects. Immunofluorescence wasperformed to observe the expression of microfilaments and transient receptor potential melastatin 7 (TRPM7). The levels of F-actin/G-actin and osteogenesis-related proteins under LIPUS alone or LIPUS combined with cytoskeleton interfering drugs (Cytochalasin D [CytoD] or Jasplakinolide [JA]) were assayed by western blot. Quantitative real-time reverse transcription polymerase chain reaction was utilized to measure the expression of Trpm7 mRNA. Moreover, adenoviral Trpm7 knockdown wasverifiedusingwesternblot. The results demonstrated that LIPUS promoted bone formation in vivo. Under osteogenic induction in vitro, the osteogenesis of BMSCs induced by LIPUS was accompanied by the depolymerization and rearrangement of microfilaments and increased levels of TRPM7. By perturbing intracellular actin dynamics, CytoD enhanced the pro-osteogenicity of LIPUS and increased TRPM7 level, while JA inhibited the pro-osteogenicity of LIPUS and reduced TRPM7 level. Additionally, the knockdown of Trpm7 suppressed the osteogenic promotion of BMSCs induced by LIPUS. The transient depolymerization and rearrangement of the cytoskeleton microfilaments mediated by LIPUS can affect TRPM7 expression and subsequently promote the osteogenesis of BMSCs. This study provides further direction for exploring the molecular mechanism of LIPUS, as a mechanical stress, in facilitating the osteogenic differentiation of BMSCs.

ETV5 Silencing Produces Mesenchymal to Epithelial Transition in INS-1 (832/13) Cell Line.

ETV5 has been described to be involved in the epithelial to mesenchymal transition (EMT) mainly in cancer. It is known that EMT provokes cytoskeleton remodeling, improving cellular migratory, and invasive capabilities. Moreover, overexpression of ETV5 has been correlated to cancer development and this gene has been implicated in cell proliferation. However, little is known about the downregulation of ETV5 expression in a pancreatic cell line and the inverse mesenchymal to epithelial transition (MET). Therefore, we studied the implications of ETV5 silencing over the phenotype of the insulinoma INS-1 (832/13) cell line and described the MET by partial ETV5 silencing in the INS-1 (832/13) cell line. The downregulation of ETV5 expression was obtained by using ETV5 siRNA in the insulinoma rat cell line, INS-1 (832/13). Then, ETV5 knockdown provoked a MET phenotype observed by crystal violet staining and verified by immunohistochemistry against E-cadherin. Wound healing assay showed no migration, and F-actin stain revealed rearrangement of actin microfilaments. In addition, TGFβ1 and TGFβ3 were downregulated in the absence of ETV5. ETV5 silencing induces epithelial phenotype by downregulating TGFβ1 and TGFβ3 in INS-1 (832/13) cell line.

A protein complex of LCN2, LOXL2 and MMP9 facilitates tumor metastasis in esophageal cancer.

During malignant tumor development, the extracellular matrix (ECM) is usually abnormally regulated. Dysregulated expression of lysyl oxidase-like 2 (LOXL2), matrix metalloproteinase 9 (MMP9) and lipocalin 2 (LCN2) are associated with ECM remodeling. In this study, protein-protein interaction assays indicated that LCN2 and LOXL2 interactions and LCN2 and MMP9 interactions occurred both intracellularly and extracellularly, but interactions between LOXL2 and MMP9 only occurred intracellularly. The LCN2/LOXL2/MMP9 ternary complex promoted migration and invasion of esophageal squamous cell carcinoma (ESCC) cells, as well as tumor growth and malignant progression in vivo, while the iron chelator deferoxamine mesylate (DFOM) inhibited ESCC tumor growth. Co-overexpression of LCN2, LOXL2 and MMP9 enhanced the ability of tumor cells to degrade fibronectin and Matrigel, increased the formation and extension of filopodia, and promoted the rearrangement of microfilaments through upregulation of profilin 1. In addition, the LCN2/LOXL2/MMP9 ternary complex promoted the expression of testican-1 (SPOCK1), and abnormally activated the FAK/AKT/GSK3β signaling pathway. In summary, the LCN2/LOXL2/MMP9 ternary complex promoted the migration and invasion of cancer cells and malignant tumor progression through multiple mechanisms, and could be a potential therapeutic target.

Homo- and Heteroassociations Drive Activation of ErbB3

Dimerization or the formation of higher-order oligomers is required for the activation of ErbB receptor tyrosine kinases. The heregulin (HRG) receptor, ErbB3, must heterodimerize with other members of the family, preferentially ErbB2, to form a functional signal transducing complex. Here, we applied single molecule imaging capable of detecting long-lived and mobile associations to measure their stoichiometry and mobility and analyzed data from experiments globally, taking the different lateral mobility of monomeric and dimeric molecular species into account. Although ErbB3 was largely monomeric in the absence of stimulation and ErbB2 co-expression, a small fraction was present as constitutive homodimers exhibiting a ∼40% lower mobility than monomers. HRG stimulation increased the homodimeric fraction of ErbB3 significantly and reduced the mobility of homodimers fourfold compared to constitutive homodimers. Expression of ErbB2 elevated the homodimeric fraction of ErbB3 even in unstimulated cells and induced a ∼2-fold reduction in the lateral mobility of ErbB3 homodimers. The mobility of ErbB2 was significantly lower than that of ErbB3, and HRG induced a less pronounced decrease in the diffusion coefficient of all ErbB2 molecules and ErbB3/ErbB2 heterodimers than in the mobility of ErbB3. The slower diffusion of ErbB2 compared to ErbB3 was abolished by depolymerizing actin filaments, whereas ErbB2 expression induced a substantial rearrangement of microfilaments, implying a bidirectional interaction between ErbB2 and actin. HRG stimulation of cells co-expressing ErbB3 and ErbB2 led to the formation of ErbB3 homodimers and ErbB3/ErbB2 heterodimers in a competitive fashion. Although pertuzumab, an antibody binding to the dimerization arm of ErbB2, completely abolished the formation of constitutive and HRG-induced ErbB3/ErbB2 heterodimers, it only slightly blocked ErbB3 homodimerization. The results imply that a dynamic equilibrium exists between constitutive and ligand-induced homo- and heterodimers capable of shaping transmembrane signaling.

Rearrangement of Actin Microfilaments in the Development of Olfactory Receptor Cells in Fish

At present, it remains poorly understood how the olfactory neuron migrates through the thick neuroepithelium during its maturation from a stem cell and how it develops a specific sensitivity to environmental odorants after maturation. We investigated the cytochemical features associated with the development of olfactory cells before and after the incorporation of dendrites into the surface of the olfactory epithelium. Using cytochemical staining for the actin cytoskeleton and other cell components, we found that immature neurons acquire a streamlined shape that resembles a «hot-dog» during their migration: a dense layer of actin microfilaments forms beneath the surface membrane of the growing dendrite, and the bulk of the nuclear material moves inside this layer. We have found that when the cell makes contact with its environment, the dendritic terminal develops a wide actin layer, inside which a pore is formed. It is assumed that the functional receptors of odorants generate across this pore the first intracellular signal from environmental water-soluble odorants. These data illustrate the important role of the cytoskeleton in the differentiation of olfactory cells.

Cyclic stretch-induced the cytoskeleton rearrangement and gene expression of cytoskeletal regulators in human periodontal ligament cells

Objective: This study aimed to explore the mechanism of the stretch-induced cell realignment and cytoskeletal rearrangement by identifying several mechanoresponsive genes related to cytoskeletal regulators in human PDL cells.Material and methods: After the cells were stretched by 1, 10 and 20% strains for 0.5, 1, 2, 4, 6, 12 or 24 h, the changes of the morphology and content of microfilaments were recorded and calculated. Meanwhile, the expression of 84 key genes encoding cytoskeletal regulators after 6 and 24 h stretches with 20% strain was detected by using real-time PCR array. Western blot was applied to identify the protein expression level of several cytoskeletal regulators encoded by these differentially expressed genes.Results: The confocal fluorescent staining results confirmed that stretch-induced realignment of cells and rearrangement of microfilaments. Among the 84 genes screened, one gene was up-regulated while two genes were down-regulated after 6 h stretch. Meanwhile, three genes were up-regulated while two genes were down-regulated after 24 h stretch. These genes displaying differential expression included genes regulating polymerization/depolymerization of microfilaments (CDC42EP2, FNBP1L, NCK2, PIKFYVE, WASL), polymerization/depolymerization of microtubules (STMN1), interacting between microfilaments and microtubules (MACF1), as well as a phosphatase (PPP1R12B). Among the proteins encoded by these genes, the protein expression level of Cdc42 effector protein-2 (encoded by CDC42EP2) and Stathmin-1 (encoded by STMN1) was down-regulated, while the protein expression level of N-WASP (encoded by WASL) was up-regulated.Conclusion: The present study confirmed the cyclic stretch-induced cellular realignment and rearrangement of microfilaments in the human PDL cells and indicated several force-sensitive genes with regard to cytoskeletal regulators.

Apoptosis-linked gene 2 promotes breast cancer growth and metastasis by regulating the cytoskeleton.

Breast cancer is the most prevalent cancer in women. Although it begins as local disease, breast cancer frequently metastasizes to the lymph nodes and distant organs. Therefore, novel therapeutic targets are needed for the management of this disease. Apoptosis-linked gene 2 (ALG-2) is a calcium-binding protein crucial for diverse physiological processes and has recently been implicated in cancer development. However, it remains unclear whether this protein is involved in the pathogenesis of breast cancer. Here, we demonstrate that the expression of ALG-2 is significantly upregulated in breast cancer tissues and is correlated with clinicopathological characteristics indicative of tumor malignancy. Our data further show that ALG-2 stimulates breast cancer growth and metastasis in mice. ALG-2 also promotes breast cancer cell proliferation, survival, and motility in vitro. Mechanistic data reveal that ALG-2 disrupts the localization of centrosome proteins, resulting in spindle multipolarity and chromosome missegregation. In addition, ALG-2 drives the polarization and migration of breast cancer cells by facilitating the rearrangement of microtubules and microfilaments. These findings reveal a critical role for ALG-2 in the pathogenesis of breast cancer and have important implications for its diagnosis and therapy.

Plant acoustics: in the search of a sound mechanism for sound signaling in plants.

Being sessile, plants continuously deal with their dynamic and complex surroundings, identifying important cues and reacting with appropriate responses. Consequently, the sensitivity of plants has evolved to perceive a myriad of external stimuli, which ultimately ensures their successful survival. Research over past centuries has established that plants respond to environmental factors such as light, temperature, moisture, and mechanical perturbations (e.g. wind, rain, touch, etc.) by suitably modulating their growth and development. However, sound vibrations (SVs) as a stimulus have only started receiving attention relatively recently. SVs have been shown to increase the yields of several crops and strengthen plant immunity against pathogens. These vibrations can also prime the plants so as to make them more tolerant to impending drought. Plants can recognize the chewing sounds of insect larvae and the buzz of a pollinating bee, and respond accordingly. It is thus plausible that SVs may serve as a long-range stimulus that evokes ecologically relevant signaling mechanisms in plants. Studies have suggested that SVs increase the transcription of certain genes, soluble protein content, and support enhanced growth and development in plants. At the cellular level, SVs can change the secondary structure of plasma membrane proteins, affect microfilament rearrangements, produce Ca(2+) signatures, cause increases in protein kinases, protective enzymes, peroxidases, antioxidant enzymes, amylase, H(+)-ATPase / K(+) channel activities, and enhance levels of polyamines, soluble sugars and auxin. In this paper, we propose a signaling model to account for the molecular episodes that SVs induce within the cell, and in so doing we uncover a number of interesting questions that need to be addressed by future research in plant acoustics.

Role of Endothelial Cell Septin 7 in the Endocytosis of Candida albicans

ABSTRACTCandida albicans invades endothelial cells by binding to N-cadherin and other cell surface receptors. This binding induces rearrangement of endothelial cell actin microfilaments, which results in the formation of pseudopods that surround the organism and pull it into the endothelial cell. Here, we investigated the role of endothelial cell septin 7 (SEPT7) in the endocytosis of C. albicans hyphae. Using confocal microscopy, we determined that SEPT7 accumulated with N-cadherin and actin microfilaments around C. albicans as it was endocytosed by endothelial cells. Affinity purification studies indicated that a complex containing N-cadherin and SEPT7 was recruited by C. albicans and that formation of this complex around C. albicans was mediated by the fungal Als3 and Ssa1 invasins. Knockdown of N-cadherin by small interfering RNA (siRNA) reduced recruitment of SEPT7 to C. albicans, suggesting that N-cadherin functions as a link between SEPT7 and the fungus. Also, depolymerization of actin microfilaments with cytochalasin D decreased the association between SEPT7 and N-cadherin and inhibited recruitment of both SEPT7 and N-cadherin to C. albicans, indicating the necessity of an intact cytoskeleton in the functional interaction between SEPT7 and N-cadherin. Importantly, knockdown of SEPT7 decreased accumulation of N-cadherin around C. albicans in intact endothelial cells and reduced binding of N-cadherin to this organism, as revealed by the affinity purification assay. Furthermore, SEPT7 knockdown significantly inhibited the endocytosis of C. albicans. Therefore, in response to C. albicans infection, SEPT7 forms a complex with endothelial cell N-cadherin, is required for normal accumulation of N-cadherin around C. albicans hyphae, and is necessary for maximal endocytosis of the organism.

Adhesion and invasion to duck embryo fibroblast cells by Riemerella anatipestifer

Here, we investigated adhesion and invasion of Riemerella anatipestifer (RA) to primary duck embryo fibroblast (DEF) cells. The ability of RA to adhere to, and more importantly, to invade DEF cells was demonstrated by using a gentamicin invasion assay and was confirmed by transmission electron microscopy (TEM). Adhesion of RA could be found by TEM after 1 h of inoculation. Both apoptosis and necrocytosis of DEF were indicated by TEM after 10 h of incubation, which suggested a complex mechanism of DEF cell death induced by RA. Our results showed that internalized RA had the ability to leave the DEF cells. Inhibition studies indicated that RA proteins play a role in adhesion. Moreover, invasion of RA to DEF cells was shown to require rearrangement of actin microfilaments and microtubular cytoskeletal elements. Because the adhesion and invasion ability of RA to DEF cells could be demonstrated in vitro, similar processes might occur in vivo, where DEF cells play a crucial role in the diffusion of RA in ducks.

The Purified and Recombinant Legionella pneumophila Chaperonin Alters Mitochondrial Trafficking and Microfilament Organization

A portion of the total cellular pool of the Legionella pneumophila chaperonin, HtpB, is found on the bacterial cell surface, where it can mediate invasion of nonphagocytic cells. HtpB continues to be abundantly produced and released by internalized L. pneumophila and may thus have postinvasion functions. We used here two functional models (protein-coated beads and expression of recombinant proteins in CHO cells) to investigate the competence of HtpB in mimicking early intracellular trafficking events of L. pneumophila, including the recruitment of mitochondria, cytoskeletal alterations, the inhibition of phagosome-lysosome fusion, and association with the endoplasmic reticulum. Microscopy and flow cytometry studies indicated that HtpB-coated beads recruited mitochondria in CHO cells and U937-derived macrophages and induced transient changes in the organization of actin microfilaments in CHO cells. Ectopic expression of HtpB in the cytoplasm of transfected CHO cells also led to modifications in actin microfilaments similar to those produced by HtpB-coated beads but did not change the distribution of mitochondria. Association of phagosomes containing HtpB-coated beads with the endoplasmic reticulum was not consistently detected by either fluorescence or electron microscopy studies, and only a modest delay in the fusion of TrOv-labeled lysosomes with phagosomes containing HtpB-coated beads was observed. HtpB is the first Legionella protein and the first chaperonin shown to, by means of our functional models, induce mitochondrial recruitment and microfilament rearrangements, two postinternalization events that typify the early trafficking of virulent L. pneumophila.

Melatonin modulates microfilament phenotypes in epithelial cells: implications for adhesion and inhibition of cancer cell migration.

Cell migration and adhesion are cytoskeleton- dependent functions that play a key role in epithelial physiology. Specialized epithelial cells in water transport have specific microfilament rearrangements that make these cells adopt a polyhedral shape, forming a sealed monolayer which functions as permeability barrier. Also, specific polarized microfilament phenotypes are formed at the front and the rear of migratory epithelial cells. In pathological processes such as cancer, increased migration occurs in invasive cells driven by the formation of polarized and differential microfilament phenotypes. Melatonin, the main product secreted by the pineal gland during dark phase of the photoperiod, acts as a cytoskeletal modulator in normal and cancer cells. In this paper we will summarize evidence supporting that melatonin acts as a microfilament modulator in epithelial MDCK cells, and we will describe its effects on cytoskeleton organization involved in the mechanism by which melatonin synchronizes water transport. In addition, we will review recent data that indicate that melatonin is able to switch microfilament phenotypes in MCF-7 human mammary cancer cells, from invasive migratory cells to dormant microfilament phenotypes that occur in non- migratory cells. Moreover, we will discuss the implications of the cytoskeleton as therapeutic target for cancer cells.

Participation of Rho, ROCK‐2, and GAP activities during actin microfilament rearrangements in Entamoeba histolytica induced by fibronectin signaling

In Entamoeba histolytica little is known about the microfilament rearrangements formed by actin and ABPs. Fibronectin regulates many aspects of cell behavior involving the actin cytoskeleton and members of the Rho family of small GTPases. Using trophozoites interacted with fibronectin and glass, we present evidence related with the formation and regulation of different microfilament rearrangements and their cellular distribution, the effect of actin affecting drugs on these arrangements, and on trophozoites adhesion; we also demonstrate that actin isoforms are induced after adhesion, and also the selective participation of specific actin binding proteins such as ABP-120 and phospho-paxillin, regarding their location in the different actin structures. In addition, we show results that confirm the participation of EhRho, ROCK-2, and GAP activities. We propose that fibronectin induced signaling in E. histolytica trophozoites have important consequences in the actin cytoskeleton that may affect its behavior during the invasive process in the host.

Leptin as an immunological adjuvant: enhanced migratory and CD8+T cell stimulatory capacity of human dendritic cells exposed to leptin

Leptin is an adipocyte-derived hormone/cytokine that links nutrition, metabolism, and immune homeostasis and is endowed to modulate several immune responses. We previously demonstrated that both immature and mature human dendritic cells (DCs) express a functional leptin receptor, and we found that leptin activates DCs, licenses them for Th1 priming, and promotes DC survival. Moreover, we found that leptin induces rearrangement of actin microfilaments, leading to uropod and ruffle formation. Here we monitor the effects of leptin on DC migratory capacities, focusing on the intracellular signaling driving cytoskeleton rearrangement. We found that leptin increases immature DC migratory performance both by favoring cytoskeleton dynamics and by up-regulating CCR7 surface expression, thus favoring chemotactic responsiveness. We found that in immature DCs, leptin activates cofilin, favoring the turnover of actin microfilaments, and, by triggering Vav phosphorylation, promotes Rac1 activation. Finally, we found that in immature DCs, leptin up-regulates interleukin-12p70 production on CD40 stimulation and, more importantly, increases their capacity to stimulate activation of autologous CD8(+) T cells. Taken altogether, the findings herein highlight the potential use of leptin as an adjuvant tool in vaccination protocols employing ex vivo-generated autologous DCs.

Melatonin induces neuritogenesis at early stages in N1E‐115 cells through actin rearrangements via activation of protein kinase C and Rho‐associated kinase

Melatonin increases neurite formation in N1E-115 cells through microtubule enlargement elicited by calmodulin antagonism and vimentin intermediate filament reorganization caused by protein kinase C (PKC) activation. Microfilament rearrangement is also a necessary process in growth cone formation during neurite outgrowth. In this work, we studied the effect of melatonin on microfilament rearrangements present at early stages of neurite formation and the possible participation of PKC and the Rho-associated kinase (ROCK), which is a downstream kinase in the PKC signaling pathway. The results showed that 1 nm melatonin increased both the number of cells with filopodia and with long neurites. Similar results were obtained with the PKC activator phorbol 12-myristate 13-acetate (PMA). Both melatonin and PMA increased the quantity of filamentous actin. In contrast, the PKC inhibitor bisindolylmaleimide abolished microfilament organization elicited by either melatonin or PMA, while the Rho inhibitor C3, or the ROCK inhibitor Y27632, abolished the bipolar neurite morphology of N1E-115 cells. Instead, these inhibitors prompted neurite ramification. ROCK activity measured in whole cell extracts and in N1E-115 cells was increased in the presence of melatonin and PMA. The results indicate that melatonin increases the number of cells with immature neurites and suggest that these neurites can be susceptible to differentiation by incoming extracellular signals. Data also indicate that PKC and ROCK are involved at initial stages of neurite formation in the mechanism by which melatonin recruits cells for later differentiation.

Re‐organization of the cytoskeleton and endoplasmic reticulum in the Arabidopsis pen1‐1 mutant inoculated with the non‐adapted powdery mildew pathogen, Blumeria graminis f. sp. hordei

SUMMARY Plant cells attacked by microorganisms rapidly translocate cytoplasm to the site of pathogen penetration, a response that usually involves rearrangement of actin microfilaments. In this study, we monitored re-organization of green fluorescent protein (GFP)-labelled actin microfilaments, microtubules and endoplasmic reticulum (ER) during infection by the powdery mildew pathogen Blumeria graminis f. sp. hordei in non-host Arabidopsis and in the Arabidopsis penetration 1-1 (pen1-1) mutant, which shows increased penetration susceptibility to non-adapted pathogens. Comparison of pen1-1 with wild-type Arabidopsis showed that the actin, microtubule and ER networks all responded in the pen1-1 mutant as they do in wild-type plants. Actin microfilaments became focused on the penetration site and ER accumulated at the penetration site while the overall arrangement of microtubule arrays was largely unaffected. These results indicate that the block in vesicle secretion conferred by the pen1-1 mutation does not interfere with cytoplasmic aggregation or recruitment of actin or ER to the infection site. In the pen1-1 mutant, the higher rate of successful penetration by the non-adapted pathogen results in an increased incidence of hypersensitive cell death. In dying cells, the structure of the ER was rapidly destroyed, in contrast to actin microfilaments and microtubules which remained for a longer time after the initiation of cell death. In Arabidopsis with GFP-tagged tubulin, fluorescent vesicle-like structures appeared near the cell surface during the initiation of cell death. In both wild-type and pen1-1 mutant plants, in cells surrounding the dying cell, bundles of actin microfilaments focused on the anticlinal walls adjacent to the dead cell and ER accumulated in the cortical cytoplasm near the dead cell. These observations suggest that the neighbouring, non-infected cells use actin-based transport to secrete material at the surface adjacent to the dead cell. They also indicate that disruption of secretion (in the pen1-1 mutant) does not inhibit transmission of signals from the dying cells to their neighbours.

A cell-penetrating peptide derived from mammalian cell uptake protein of Mycobacterium tuberculosis

A Mycobacterium tuberculosis membrane protein called Mycobacterium cell entry protein (Mce1A) was previously shown to mediate the uptake of nonpathogenic Escherichia coli and latex beads by nonphagocytic mammalian cells. Here we characterize further the in vitro invasive activity of Mce1A using colloidal gold nanoparticles and fluorescent latex microspheres. Mce1A-coated colloidal gold particles induced plasma membrane invagination and entered membrane-bound compartments inside HeLa cells. Few of the protein-coated particles were also found in the cytosol compartment. Cytochalasin D and nocodazole inhibited the uptake by HeLa cells, indicating that rearrangement of both microtubules and microfilaments was necessary for the uptake. The functional domain of Mce1A for invasion was narrowed to a highly basic 22-amino acid sequence termed Inv3. A synthetic Inv3 peptide stimulated uptake of colloidal gold particles as well as latex microspheres by HeLa cells. A chimeric protein composed of Inv3 sequence at the N terminus of β-galactosidase appeared to stain the nuclear membrane, suggesting that it entered the HeLa cell cytoplasm. These observations suggest that the cell uptake activity of Mce1A is confined to a small peptide domain located in the core region of the protein. Inv3 could be used to ferry any protein in fusion with it into mammalian cells and may serve as a potent nonviral delivery system.

Haemophilus parasuis invades porcine brain microvascular endothelial cells

Haemophilus parasuis, an important swine pathogen, is the aetiological agent of Glässer's disease. It is responsible for cases of polyserositis, meningitis and pneumonia in young pigs. To date, 15 serotypes have been described, although several non-typable isolates are frequently recovered from diseased animals. The pathogenesis of H. parasuis infection is poorly understood. To cause meningitis, H. parasuis would have to cross the blood-brain barrier (BBB), composed of brain microvascular endothelial cells (BMEC). The objective of this study was to investigate the ability of H. parasuis to interact with porcine brain microvascular endothelial cells (PBMEC). It was demonstrated that the serotype 5 reference strain of H. parasuis, Nagasaki (originally recovered from a case of meningitis), was able to adhere at very high levels to and, most importantly, invade PBMEC. These capacities were confirmed by electron microscopy. Actinobacillus pleuropnemoniae serotype 7 (strain WF 83), used as negative control, was not able to adhere to or invade PBMEC. Comparisons of the levels of adhesion and invasion by several H. parasuis field strains from different serotypes isolated from cases of either meningitis or pneumonia showed that isolates of serotypes 4 and 5 had a higher invasion capacity than isolates belonging to other serotypes. Inhibition studies demonstrated that PBMEC invasion by H. parasuis required rearrangement of actin microfilaments and microtubular cytoskeletal elements but not active bacterial DNA, RNA or protein synthesis. Characterization studies demonstrated that proteinaceous invasin(s) does not seem to play a major role in entry of H. parasuis into PBMEC. Intracellular viable H. parasuis were found in PBMEC up to 6 h after antibiotic treatment. Even at high bacterial doses, H. parasuis was not toxic to PBMEC. In swine, the invasion of endothelial cells of the BBB may play an important role in the pathogenesis of meningitis caused by H. parasuis.

Molecular Mechanism of Nrf2 Activation by Oxidative Stress

The capacity of cells to maintain homeostasis during oxidative stress resides in activation or induction of protective enzymes. Nuclear-factor-E2-related factor (Nrf)-2 as a member of bZIP transcription factors is expressed in a variety of tissues. Transcriptional activation of antioxidant genes through an antioxidant response element (ARE) is largely dependent upon Nrf2. The genes that contain a functional ARE include those encoding GSTA1, GSTA2, NAD(P)H:quinone reductase, and gamma-glutamylcysteine synthetase heavy and light subunits that play a role in defense against oxidative stress. Previously, we showed that phosphatidylinositol 3-kinase (PI3-kinase) controls nuclear translocation of Nrf2 in response to oxidative stress, which involves rearrangement of actin microfilaments. Now, we report that PI3-kinase is responsible for the rise of cellular Ca(2+), which is requisite for nuclear translocation of Nrf2. Immunocytochemistry and subcellular fractionation analyses revealed that Nrf2 relocated from the cytoplasm to the plasma membrane prior to its nuclear translocation. We further found that CCAAT/enhancer binding protein-beta (C/EBPbeta), peroxisome proliferatoractivated receptor-gamma (PPARgamma), and retinoid X receptor (RXR) heterodimer serve as the activating transcription factors for the phase II gene induction. Hence, PI3-kinase-mediated Nrf2 activation in combination with activating PPARgamma-RXR and C/EBPbeta contributes to antioxidant phase II enzyme induction via coordinate gene transactivation.

Melatonin as a cytoskeletal modulator: implications for cell physiology and disease

The cytoskeleton is a phylogenetically well-preserved structure that plays a key role in cell physiology. Dynamic and differential changes in cytoskeletal organization occur in cellular processes according to the cell type and the specific function. In neurons, microtubules, microfilaments and intermediate filament (IF) rearrangements occur during axogenesis, and neurite formation which eventually differentiate into axons and dendrites to constitute synaptic patterns of connectivity. In epithelial cells, dynamic modifications occur in the three main cytoskeletal components and phosphorylation of cytoskeletal associated proteins takes place during the formation of the epithelial cell monolayer that eventually will transport water. In pathological processes such as neurodegenerative and psychiatric diseases an abnormal cytoskeletal organization occurs. Melatonin, the main product secreted by pineal gland during dark phase of the photoperiod, is capable of influencing microfilament, microtubule and IF organization by acting as a cytoskeletal modulator. In this paper we will summarize the evidence which provides the data that melatonin regulates cytoskeletal organization and we describe recent findings, which indicate that melatonin effects on microfilament rearrangements in stress fibers are involved in the mechanism by which the indole synchronizes water transport in kidney-derived epithelial cells. In addition, we review recent data, which indicates that melatonin protects the neuro-cytoskeletal organization from damage caused by free radicals contributing to cell survival, in addition to the already described mechanism elicited by the indole to prevent apoptosis and to scavenge free radicals. Moreover, we discuss the implications of an altered cytoskeletal organization for neurodegenerative and psychiatric illnesses and its re-establishment by melatonin.