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Shank3, a key molecule related to the development and deterioration of autism, has recently been found to downregulate in the murine brain after ischemia/reperfusion (I/R). Despite this discovery, however, its effects on neuronal injury and the mechanism underlying the effects remain to be clarified. To address this, in this study, based on genetically modified mice models, we revealed that the expression of Shank3 showed a time-dependent change in murine hippocampal neurons after I/R, and that conditional knockout (cko) of Shank3 in neurons resulted in aggravated neuronal injuries. The protective effects of Shank3 against oxidative stress and inflammation after I/R were achieved through direct binding STIM1 and subsequent proteasome-mediated degradation of STIM1. The STIM1 downregulation induced the phosphorylation of downstream Nrf2 Ser40, which subsequently translocated to the nucleus, and further increased the expression of antioxidant genes such as NQO1 and HO-1 in HT22 cells. In vivo, the study has further confirmed that double knockout of Shank3 and Stim1 alleviated oxidative stress and inflammation after I/R in Shank3 cko mice. In conclusion, the present study has demonstrated that Shank3 interacts with STIM1 and inhibits post-I/R neuronal oxidative stress and inflammatory response via the Nrf2 pathway. This interaction can potentially contribute to the development of a promising method for I/R treatment., Competing Interests: Declaration of competing interest The authors have declared that no competing interests exist., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Prostate cancer is the second leading cause of noncutaneous cancer-related deaths in American men. Androgen deprivation therapy (ADT), radical prostatectomy, and radiotherapy remain the primary treatment for patients with early-stage prostate cancer (castration-sensitive prostate cancer). Following ADT, many patients ultimately develop metastatic castration-resistant prostate cancer (mCRPC). Standard chemotherapy options for CRPC are docetaxel (DTX) and cabazitaxel, which increase median survival, although the development of resistance is common. Cancer stem-like cells possess mesenchymal phenotypes [epithelial-to-mesenchymal transition (EMT)] and play crucial roles in tumor initiation and progression of mCRPC. We have shown that low-dose continuous administration of topotecan (METRO-TOPO) inhibits prostate cancer growth by interfering with key cancer pathway genes. This study utilized bulk and single-cell or whole-transcriptome analysis [(RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq)], and we observed greater expression of several EMT markers, including Vimentin , hyaluronan synthase-3 , S100 calcium binding protein A6 , TGFB1 , CD44 , CD55 , and CD109 in European American and African American aggressive variant prostate cancer (AVPC) subtypes-mCRPC, neuroendocrine variant (NEPC), and taxane-resistant. The taxane-resistant gene FSCN1 was also expressed highly in single-cell subclonal populations in mCRPC. Furthermore, metronomic-topotecan single agent and combinations with DTX downregulated these EMT markers as well as CD44 + and CD44 + /CD133 + "stem-like" cell populations. A microfluidic chip-based cell invasion assay revealed that METRO-TOPO treatment as a single agent or in combination with DTX was potentially effective against invasive prostate cancer spread. Our RNA-seq and scRNA-seq analysis were supported by in silico and in vitro studies, suggesting METRO-TOPO combined with DTX may inhibit oncogenic progression by reducing cancer stemness in AVPC through the inhibition of EMT markers and multiple oncogenic factors/pathways., Significance: The utilization of metronomic-like dosing regimens of topotecan alone and in combination with DTX resulted in the suppression of makers associated with EMT and stem-like cell populations in AVPC models. The identification of molecular signatures and their potential to serve as novel biomarkers for monitoring treatment efficacy and disease progression response to treatment efficacy and disease progression were achieved using bulk RNA-seq and single-cell-omics methodologies., (© 2023 The Authors; Published by the American Association for Cancer Research.)

PDZ-LIM domain-containing Protein 2 (PDLIM2) has been reported to be downregulated in ovarian cancer. However, its exact function and mechanism in regulating ovarian cancer progression have not been elucidated. This work researched the exert effect and mechanism of PDLIM2 on ovarian cancer progression. Briefly, PDLIM2 expression in clinical tissues of ovarian cancer patients and cells was investigated by qRT-PCR and Western blot. The function of PDLIM2 on the proliferation, colony formation, migration and invasion of ovarian cancer cells was explored via cell counting kit-8, colony formation and Transwell assays. To verify whether PDLIM2 regulates ovarian cancer progression via regulating the transforming growth factor-β (TGF-β)/Smad pathway, exogenous TGF-β (10 ng/mL) treatment was performed on the PDLIM2-overexpressed ovarian cancer cells. PDLIM2 effect on the in vivo growth of ovarian cancer cells was researched by establishing a xenograft tumor model. Immunohistochemistry and Western blot were performed to protein expression in cells and tissues. As a result, PDLIM2 was low-expressed in ovarian cancer tissues/cells. PDLIM2 upregulation attenuated the proliferation, colony formation, migration, invasion and epithelial-mesenchymal transition (EMT) of ovarian cancer cells, and inactivated the TGF-β/Smad pathway. The opposite results were found in the PDLIM2-silenced ovarian cancer cells. Exogenous TGF-β treatment abrogated the inhibition of PDLIM2 on the malignant behavior of ovarian cancer cells. PDLIM2 upregulation attenuated the in vivo growth and EMT of ovarian cancer cells. Thus, PDLIM2 attenuates the proliferation, migration, invasion and EMT of ovarian cancer cells via inactivating the TGF-β/Smad pathway. PDLIM2 may be a usefully target for ovarian cancer treatment., (© 2023 John Wiley & Sons Ltd.)

Background: mGlu5 metabotropic glutamate receptors are considered as candidate drug targets in the treatment of "monogenic" forms of autism spectrum disorders (ASD), such as Fragile- X syndrome (FXS). However, despite promising preclinical data, clinical trials using mGlu5 receptor antagonists to treat FXS showed no beneficial effects., Objective: Here, we studied the expression and function of mGlu5 receptors in the striatum of adult BTBR mice, which model idiopathic forms of ASD, and behavioral phenotype., Methods: Behavioral tests were associated with biochemistry analysis including qPCR and western blot for mRNA and protein expression. In vivo analysis of polyphosphoinositides hydrolysis was performed to study the mGlu5-mediated intracellular signaling in the striatum of adult BTBR mice under basal conditions and after MTEP exposure., Results: Expression of mGlu5 receptors and mGlu5 receptor-mediated polyphosphoinositides hydrolysis were considerably high in the striatum of BTBR mice, sensitive to MTEP treatment. Changes in the expression of genes encoding for proteins involved in excitatory and inhibitory neurotransmission and synaptic plasticity, including Fmr1, Dlg4, Shank3, Brd4, bdnf-exon IX, Mef2c, and Arc, GriA2, Glun1, Nr2A, and Grm1, Grm2, GriA1, and Gad1 were also found. Behaviorally, BTBR mice showed high repetitive stereotypical behaviors, including self-grooming and deficits in social interactions. Acute or repeated injections with MTEP reversed the stereotyped behavior and the social interaction deficit. Similar effects were observed with the NMDA receptor blockers MK-801 or ketamine., Conclusion: These findings support a pivotal role of mGlu5 receptor abnormal expression and function in idiopathic ASD adult forms and unveil novel potential targets for therapy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Bladder cancer, specifically, muscle-invasive bladder cancer (MIBC), is among the most common malignant tumors. Patients with MIBC who cannot tolerate standard drugs require novel treatments. Targeting apoptosis may help treat cancer, which may be achieved with the use of some natural products. Nodosin, found in Isodon serra (Maxim.) Kudo (known as Xihuangcao), may inhibit bladder cancer cells. Transcriptomics and proteomics dual-omic analyses revealed the network pharmacological mechanism: (1) blocking the S phase by up-regulating RPA2, CLSPN, MDC1, PDCD2L, and E2F6 gene expressions, suppressing cancer cell proliferation; (2) inducing apoptosis and autophagy and restraining ferroptosis by up-regulating HMOX1, G0S2, SQSTM1, FTL, SLC7A11, and AIFM2 gene expressions; (3) preventing cancer cell migration by down-regulating NEXN, LIMA1, CFL2, PALLD, and ITGA3 gene expressions. In vivo , nodosin inhibited bladder cancer cell growth in a model of xenograft tumor in nude mice. This study is the first to report basic research findings on the network pharmacological mechanism of cytotoxicity of bladder cancer cells by nodosin, providing novel evidence for the application of nodosin in the field of oncology; however, other mechanisms may be involved in the effects of nodosin for further research. These findings provide a foundation for the development of novel MIBC drugs.

Autism Spectrum Disorders (ASD) are neurodevelopmental disorders whose diagnosis relies on deficient social interaction and communication together with repetitive behavior. To date, no pharmacological treatment has been approved that ameliorates social behavior in patients with ASD. Based on the excitation/inhibition imbalance theory of autism, we hypothesized that bromide ions, long used as an antiepileptic medication, could relieve core symptoms of ASD. We evaluated the effects of chronic sodium bromide (NaBr) administration on autistic-like symptoms in three genetic mouse models of autism: Oprm1 -/- , Fmr1 -/- and Shank3 Δex13-16 -/- mice. We showed that chronic NaBr treatment relieved autistic-like behaviors in these three models. In Oprm1 -/- mice, these beneficial effects were superior to those of chronic bumetanide administration. At transcriptional level, chronic NaBr in Oprm1 null mice was associated with increased expression of genes coding for chloride ions transporters, GABA A receptor subunits, oxytocin and mGlu4 receptor. Lastly, we uncovered synergistic alleviating effects of chronic NaBr and a positive allosteric modulator (PAM) of mGlu4 receptor on autistic-like behavior in Oprm1 -/- mice. We evidenced in heterologous cells that bromide ions behave as PAMs of mGlu4, providing a molecular mechanism for such synergy. Our data reveal the therapeutic potential of bromide ions, alone or in combination with a PAM of mGlu4 receptor, for the treatment of ASDs., (© 2022. The Author(s), under exclusive licence to American College of Neuropsychopharmacology.)

Zearalenone (ZEN) is a worldwide fusarotoxin that poses a threat to the consumer due to its chronic toxicity. Herein we examined the effects of ZEN on adult mouse testis, focusing on oxidative stress, biochemical and morphological parameters. In addition, since cytoskeletal remodeling is a key event for the production of good quality gametes, the expression and localization of two proteins, Dishevelled-associated activator of morphogenesis 1 (DAAM1) and Prolyl endopeptidase (PREP), involved in cytoskeletal dynamics during spermatogenesis were evaluated. To ameliorate the testicular dysfunction induced by ZEN we tested the eventual protective effects of lactic bacteria Lactobacillus plantarum MON03 (LP) on its reprotoxicity. Adult male mice were then treated daily for 2 wks by oral gavage with ZEN and/or LP. The results confirmed that ZEN altered sperm parameters, generated oxidative stress and provoked structural alteration, evidenced by the increased number of abnormal seminiferous tubules and of apoptotic cells, particularly Leydig cells. Interestingly, at molecular level we evaluated, for the first time, the ability of ZEN to alter DAAM1 and PREP protein level and localization. Moreover, the co-treatment with LP, thanks to its capacity to reduce ZEN bioavailability in the gastrointestinal tract, ameliorated all the considered parameters. These results suggest the use of this probiotic as food supplement to prevent/counteract ZEN-induced reprotoxicity., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Intra-abdominal infection is the second most common cause of sepsis, and the mortality rate from abdominal sepsis remains high. High molecular weight (HMW) hyaluronic acid (HA) has been studied in sterile injury models as an anti-inflammatory and anti-permeability agent. This study evaluated the therapeutic effects of intraperitoneal HMW HA administration in mice with peritonitis-induced sepsis. Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP), followed 4 h later by an intraperitoneal injection of HMW HA (20 mg/kg) solution or phosphate buffered saline (PBS). Survival, physiological data, organ injury, bacterial burden, and inflammatory cytokine levels were assessed in the CLP mice. To assess the effect of HA on macrophage phagocytosis activity, RAW264.7 cells, primed with lipopolysaccharide, were exposed with either PBS or HMW HA (500 μg/mL) prior to exposure to 10 CFU of E coli bacteria. HMW HA instillation significantly improved blood oxygenation, lung histology, and survival in CLP mice. Inflammatory cytokine levels in the plasma and bacterial burdens in the lung and spleen were significantly decreased by HA administration at 24 h after CLP. At 6 h after CLP, HA significantly decreased bacterial burden in the peritoneal lavage fluid. HMW HA administration significantly increased E coli bacterial phagocytosis by RAW264.7 cells in part through increased phosphorylation of ezrin/radixin/moesin, a known downstream target of CD44 (a HA receptor); ezrin inhibition abolished the enhanced phagocytosis by RAW264.7 cells induced by HA. Intraperitoneal administration of HMW HA had therapeutic effects against CLP-induced sepsis in terms of suppressing inflammation and increasing antimicrobial activity.

Microvilli are finger-like membrane protrusions, supported by the actin cytoskeleton, and found on almost all cell types. A growing body of evidence suggests that the dynamic lymphocyte microvilli, with their highly curved membranes, play an important role in signal transduction leading to immune responses. Nevertheless, challenges in modulating local membrane curvature and monitoring the high dynamicity of microvilli hampered the investigation of the curvature-generation mechanism and its functional consequences in signaling. These technical barriers have been partially overcome by recent advancements in adapted super-resolution microscopy. Here, we review the up-to-date progress in understanding the mechanisms and functional consequences of microvillus formation in T cell signaling. We discuss how the deformation of local membranes could potentially affect the organization of signaling proteins and their biochemical activities. We propose that curved membranes, together with the underlying cytoskeleton, shape microvilli into a unique compartment that sense and process signals leading to lymphocyte activation., (Copyright © 2020 Orbach and Su.)

Endothelial progenitor cells (EPCs) are bone-marrow derived cells that are critical in the maintenance of endothelial wall integrity and protection of ischemic myocardium through the formation of new blood vessels (vasculogenesis) or proliferation of pre-existing vasculature (angiogenesis). Diabetes mellitus (DM) and the metabolic syndrome are commonly associated with ischemic heart disease through its pathological effects on the endothelium and consequent endothelial dysfunction. Thymosin-β4 (Tβ4) which expressed in the embryonic heart is critical in epicardial and coronary artery formation. In this study, we explored the effects of Tβ4 treatment on diabetic EPCs in vitro and intramyocardial injection of Tβ4-treated and non-Tβ4 treated EPCs following acute myocardial infarction (MI) of diabetic rats in vivo. It was found that 10 ng/mL Tβ4 increased migration, tubule formation, and angiogenic factor secretion of diabetic EPCs in vitro. In vivo, although implantation of Tβ4 treated diabetic EPCs significantly increased capillary density and attracted more c-Kit positive progenitor cells into the infarcted hearts as compared with implantation of non-Tβ4 treated diabetic EPCs, the significantly improved left ventricular ejection fraction was only found in the rats which received non-Tβ4 treated EPCs. The data suggests that a low dose Tβ4 increases diabetic EPC migration, tubule formation, and angiogenic factor secretion. However, it did not improve the effects of EPCs on left ventricular pump function in diabetic rats with MI.

MDR and tumor migration and invasion are still the main obstacles to effective breast cancer chemotherapies. Transgelin 2 has recently been shown to induce drug resistance, tumor migration, and invasion. The aim of this study was to determine the biological functions of Transgelin 2 and the mechanism underlying how Transgelin 2 induces paclitaxel (PTX) resistance and the migration and invasion of breast cancer. We detected that the protein level of Transgelin 2 was significantly upregulated in breast cancer tissues compared with adjacent nontumor tissues. A bioinformatics analysis indicated that Transgelin 2 was significantly related to clinicopathologic parameters and patient prognosis. Overexpression of Transgelin 2 enhanced the migration and invasion of human breast cancer cells and decreased the sensitivity of breast cancer cells to paclitaxel. Meanwhile, the tumorigenesis and metastasis of breast cancer cells were also enhanced by Transgelin 2 overexpression in vivo Moreover, Transgelin 2 overexpression activated the PI3K/Akt/GSK-3β pathway by increasing the phosphorylation levels of Akt and GSK-3β and decreasing the expression of PTEN. We also found that Transgelin 2 could directly interact with PTEN and was located upstream of PTEN. Furthermore, the PI3K/Akt pathway inhibitor MK-2206 reversed the resistance to paclitaxel and inhibited the migration and invasion of breast cancer cells. These findings indicate that Transgelin 2 promotes paclitaxel resistance and the migration and invasion of breast cancer by directly interacting with PTEN and activating the PI3K/Akt/GSK-3β pathway. Transgelin 2 may therefore be useful as a novel biomarker and therapeutic target for breast cancer., (©2019 American Association for Cancer Research.)

During the development of mammalian cortex, late neurons generated by neuronal progenitors bypass earlier-born neurons and migrate to reach upper layers of cortical plate in an inner-to-outer fashion. Filamentous-actin (F-actin) can regulate neuronal migration, whereas Coactosin-like protein 1 (Cotl1) modulates F-actin. Lys 75 and Arg 73 of Cotl1 play an important role in binding F-actin; when they are mutated to Glu, Cotl1 cannot bind F-actin, called as a non-actin-binding mutant (ABM). The Lys 131 site of Cotl1, the 5-Lipoxygenase (5LO) binding site, is spatially close to Lys 75, leading to impact the binding of Cotl1 to F-actin. When Lys 131 is mutated to Ala (K131A), Cotl1 cannot bind to 5LO. We have demonstrated that overexpression of Cotl1 inhibited neuronal migration and increased the length of neuronal leading processes. To further explore cellular and molecular mechanisms of Cotl1's effect on neuronal migration, we constructed two mutant vectors-Cotl1-ABM and Cotl1-K131A and studied using in utero electroporation and primary neuronal culture technique. Results indicated that in the Cotl1-ABM group, the neuronal migration and length of the leading process both recovered as control neurons at the postnatal day 1 (P1), while in the Cotl1-K131A group, numerous neurons remained in deeper layers of cortical plate or intermediate zone. However, at P7, most Cotl1-K131A transfected neurons reached their destination. Moreover, we found that overexpression of Cotl1 inhibited the proliferation and mitotic activity of NPs. Therefore, These results demonstrated that Cotl1 played an important role in mouse neocortical development.

Thymosin beta 4 (T β 4), an actin-sequestering protein, is involved in tissue development and regeneration. It prevents inflammation and fibrosis in several tissues. We investigated the role of T β 4 in chronic ethanol- and acute lipopolysaccharide- (LPS-) induced mouse liver injury. C57BL/6 mice were fed 5% ethanol in liquid diet for 4 weeks plus binge ethanol (5 g/kg, gavage) with or without LPS (2 mg/kg, intraperitoneal) for 6 hours. T β 4 (1 mg/kg, intraperitoneal) was administered for 1 week. We demonstrated that T β 4 prevented ethanol- and LPS-mediated increase in liver injury markers as well as changes in liver pathology. It also prevented ethanol- and LPS-mediated increase in oxidative stress by decreasing ROS and lipid peroxidation and increasing the antioxidants, reduced glutathione and manganese-dependent superoxide dismutase. It also prevented the activation of nuclear factor kappa B by blocking the phosphorylation of the inhibitory protein, I κ B, thereby prevented proinflammatory cytokine production. Moreover, T β 4 prevented fibrogenesis by suppressing the epigenetic repressor, methyl-CpG-binding protein 2, that coordinately reversed the expression of peroxisome proliferator-activated receptor- γ and downregulated fibrogenic genes, platelet-derived growth factor- β receptor, α -smooth muscle actin, collagen 1, and fibronectin, resulting in reduced fibrosis. Our data suggest that T β 4 has antioxidant, anti-inflammatory, and antifibrotic potential during alcoholic liver injury.

Background and Objective: Human periodontal ligament cells (HPDLCs) express transforming growth factor-β1 (TGF-β1) that regulates differentiation and proliferation, and plays key roles in homeostasis of PDL tissue. Transgelin is a cytoskeleton-associated protein with an Smad-binding element in its gene promoter region. In this study, we examined the localization and potential function of transgelin in PDL tissue and cells., Material and Methods: Microarray analysis of HPDLC lines (2-14, 2-23 and 2-52) was performed. Expression of transgelin in HPDLCs was examined by quantitative reverse transcription-polymerase chain reaction, immunofluorescence staining and western blot analysis. Effects of TGF-β1 and its signaling inhibitor, SB431542, on transgelin expression in HPDLCs were examined by western blot analysis. The effects of transgelin knockdown by small interfering RNA (siRNA) on HPDLC proliferation stimulated by TGF-β1 were assessed by WST-1 assay., Results: In microarray and quantitative reverse transcription-polymerase chain reaction analyses, the expression levels of transgelin (TAGLN) in 2-14 and 2-23 cells, which highly expressed PDL markers such as periostin (POSTN), tissue non-specific alkaline phosphatase (ALPL), α-smooth muscle actin (ACTA2) and type I collagen A1 (COL1A1), was significantly higher than those in 2-52 cells that expressed PDL markers weakly. Immunohistochemical and immunofluorescence staining revealed expression of transgelin in rat PDL tissue and HPDLCs. In HPDLCs, TGF-β1 treatment upregulated transgelin expression, whereas inhibition of the type 1 TGF-β1 receptor by SB431542 suppressed this upregulation. Furthermore, TAGLN siRNA transfection did not promote the proliferation of HPDLCs treated with TGF-β1. The expression levels of CCNA2 and CCNE1, which regulate DNA synthesis and mitosis through the cell cycle, were also not upregulated in HPDLCs transfected with TAGLN siRNA., Conclusion: Transgelin is expressed in PDL tissue and might have a role in HPDLC proliferation induced by TGF-β1 stimulation., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Designing bacterial vectors for cancer therapy represents a major challenge. Recent studies have explored novel strategies to balance benefit and safety. A study by Mercado-Lubo et al. has developed a next-generation concept combining bacterial properties with nanoparticles, demonstrating efficacy in combination with chemotherapeutics., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Host actin cytoskeleton polymerization has been shown to play an important role during Trypanosoma cruzi internalization into mammalian cell. The structure and dynamics of the actin cytoskeleton in cells are regulated by a vast number of actin-binding proteins. Here we aimed to verify the impact of AFAP-1L1, during invasion and multiplication of T. cruzi. Knocking-down AFAP-1L1 increased parasite cell invasion and intracellular multiplication. Thus, we have shown that the integrity of the machinery formed by AFAP-1L1 in actin cytoskeleton polymerization is important to hinder parasite infection., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Purpose: To investigate the glial response of the rat retina to single or repeated intravitreal injections (IVI)., Methods: Albino Sprague-Dawley rats received one or three (one every 7 days) IVI of anti-rat VEGF (5 μL; 0.015 μg/μL), triamcinolone (2.5 or 5 μL; 40 μg/μL; Trigón Depot), bevacizumab (5 μL; 25 μg/μL; Avastin), or their vehicles (PBS and balanced salt solution) and were processed 7 days after the last injection. Retinas were dissected as whole mounts and incubated with antibodies against: Iba1 (Ionized Calcium-Binding Adapter Molecule 1) to label retinal microglia, GFAP (Glial Fibrillary Acidic Protein) to label macroglial cells, and vimentin to label Müller cells. The retinas were examined with fluorescence and confocal microscopy, and the numbers of microglial cells in the inner retinal layers were quantified using a semiautomatic method., Results: All the injected substances caused an important micro- and macroglial response locally at the injection site and all throughout the injected retina that was exacerbated by repeated injections. The microglial response was also observed but was milder in the contralateral noninjected eyes. The IVI of the humanized antibody bevacizumab caused a very strong microglial reaction in the ipsilateral retina. Two types of macroglial response were observed: astrocyte hypertrophy and Müller end-foot hypertrophy. While astrocyte hypertrophy was widespread throughout the injected retina, Müller end-foot hypertrophy was localized and more extensive with triamcinolone use or after repeated injections., Conclusions: Intravitreal injections cause micro- and macroglial responses that vary depending on the injected agent but increase with repeated injections. This inflammatory glial response may influence the effects of the injected substances on the retina.

The goal of this work was to elucidate the mechanism of inhibition of the actin-activated ATPase of myosin subfragment-1 (S1) by the calponin-like protein from mussel bivalve muscle. The calponin-like protein (Cap) is a 40-kDa actin-binding protein from the bivalve muscle of the mussel Crenomytilus grayanus. Kinetic parameters Vmax and KATPase of actomyosin ATPase in the absence and the presence of Cap were determined to investigate the mechanism of inhibition. It was found that Cap mainly causes increase in KATPase value and to a lesser extent the decrease in Vmax, which indicates that it is most likely a competitive inhibitor of actomyosin ATPase. Analysis of Vmax and KATPase parameters in the presence of tropomyosin revealed that the latter is a noncompetitive inhibitor of the actomyosin ATPase.

Background: Ticagrelor is a P2Y12 receptor antagonist, with superior effects but also ensuing enhanced bleeding risk as compared to clopidogrel. Determination of platelet inhibition may be useful to confirm efficient platelet inhibition on an individual patient level and to identify patients at risk for bleeding. The vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation assay specifically measures platelet P2Y12 inhibition, but has so far required individual sample processing. A new ELISA-based VASP assay has been developed, which allows batch analysis after initial platelet activation. Because of the reversible binding of ticagrelor it is unclear if the ELISA and flow cytometric assays provide comparable results; several washing steps of the ELISA may potentially result in false low results through dilution., Methods: We hypothesized that the conventional and new methods may be comparable when ticagrelor is used. We pair-wise compared the platelet reactivity index (PRI) between assays in a prospective clinical trial. Six healthy volunteers received a single 180 mg loading dose of ticagrelor., Results: PRI-values of the two methods correlated well (r = 0.97, P < 0.001). Ticagrelor rapidly decreased PRI values on average after 50 min, but nadir levels 2-6 h after ticagrelor intake were 15% higher when PRI% was measured with the flow cytometric method. Bland-Altman analysis showed that the flow cytometric assay measured markedly higher PRI levels than the new ELISA-based technique (mean difference 13%)., Conclusions: The new ELISA-based VASP assay offers an alternative to the currently used flow cytometric method, but measures lower PRI levels, particularly when PRI falls below 20% after ticagrelor intake., (© 2013 Clinical Cytometry Society.)

Background: Random skin flaps can be used throughout the hands and fingers. Thymosin β4 can increase blood flow and reduce ischemia-reperfusion injury; the study was undertaken to investigate the effect of thymosin β4 on the survival of random skin flaps., Methods: A total of 45 male Sprague-Dawley rats were used and subjected to a random-pattern skin flaps operation. Rats were randomly divided into three groups: a control group (group A: intraperitoneal injection of saline, 5 mg/kg/d) and two treatment groups (group B: intraperitoneal injection of thymosin β4, a single 5 mg/kg dose per day) and (group C: intraperitoneal injection of thymosin β4, 5 mg/kg dose twice per day). The flap surviving area was measured after 7 days, and tissue samples were stained with hematoxylin and eosin. Vascular endothelial growth factor (VEGF) expression was determined using immunohistochemical methods. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were examined with kits., Results: Thymosin β4 significantly reduced the necrotic area in the treatment groups after 7 days compared with the control group, and the rats receiving thymosin β4 5 mg/kg twice per day had the highest survival rate. VEGF expression and SOD activity markedly increased in the treatment groups compared with the control group, whereas MDA levels were lower in the treatment groups than in the control group., Conclusion: Thymosin β4 may have a dose-dependent effect to promote the survival of random skin flaps., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

In somatic cells, the position of the cell centroid is dictated by the centrosome. The centrosome is instrumental in nucleus positioning, the two structures being physically connected. Mouse oocytes have no centrosomes, yet harbour centrally located nuclei. We demonstrate how oocytes define their geometric centre in the absence of centrosomes. Using live imaging of oocytes, knockout for the formin 2 actin nucleator, with off-centred nuclei, together with optical trapping and modelling, we discover an unprecedented mode of nucleus positioning. We document how active diffusion of actin-coated vesicles, driven by myosin Vb, generates a pressure gradient and a propulsion force sufficient to move the oocyte nucleus. It promotes fluidization of the cytoplasm, contributing to nucleus directional movement towards the centre. Our results highlight the potential of active diffusion, a prominent source of intracellular transport, able to move large organelles such as nuclei, providing in vivo evidence of its biological function.

Objective: Edema due to capillary leak is a generalized and life threatening event in sepsis and major burns for which there is no causal treatment. Local burn wounds are an ideal model to investigate the impact of a new therapeutic agent on edema formation. We aimed to identify peptide sequences of cingulin that can attenuate stress-induced endothelial cytoskeleton disarrangement in vitro and which reduce burn-induced edema in vivo., Methods: Cingulin-derived peptides were screened in high content cell culture assays monitoring actin displacement and endothelial cell/cell contacts. The ears of male hairless mice (n=44) were inflicted with full thickness burns using a hot air jet. Mice with and without burn injuries were treated with Xib13 or solvent by continuous intraperitoneal application for 3 days. Edema, microcirculation, leukocyte-endothelial interactions and angiogenesis - measured as non-perfused area - were investigated over a 12-day period using intravital fluorescence microscopy., Results: Xib13 reduced endothelial stress formation and stabilized endothelial tight junctions in cell-cultures. In the burn model, Xib13 improved angiogenesis compared to controls (non-perfused area on day 12: 5.7±1.5% vs. 12.0±2.1%; p<0.05). Edema was significantly reduced at all observation points in Xib13-treated animals as compared to controls (day 12: 67.6±2.6% vs. 83.2±6.4%)., Conclusion: Xib13 improved angiogenesis, reduced edema formation and showed no side effects on other physiological parameters. Since edema formation is a serious parameter for burn conversion and is associated with survival it could provide a new treatment option for patients with burn injuries., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Microtubule end-binding (EB) proteins influence microtubule dynamic instability, a process that is essential for microtubule reorganisation during apico-basal epithelial differentiation. Here, we establish for the first time that expression of EB2, but not that of EB1, is crucial for initial microtubule reorganisation during apico-basal epithelial differentiation, and that EB2 downregulation promotes bundle formation. EB2 siRNA knockdown during early stages of apico-basal differentiation prevented microtubule reorganisation, whereas its downregulation at later stages promoted microtubule stability and bundle formation. Interestingly, although EB1 is not essential for microtubule reorganisation, its knockdown prevented apico-basal bundle formation and epithelial elongation. siRNA depletion of EB2 in undifferentiated epithelial cells induced the formation of straight, less dynamic microtubules with EB1 and ACF7 lattice association and co-alignment with actin filaments, a phenotype that could be rescued by inhibition with formin. Importantly, in situ inner ear and intestinal crypt epithelial tissue revealed direct correlations between a low level of EB2 expression and the presence of apico-basal microtubule bundles, which were absent where EB2 was elevated. EB2 is evidently important for initial microtubule reorganisation during epithelial polarisation, whereas its downregulation facilitates EB1 and ACF7 microtubule lattice association, microtubule-actin filament co-alignment and bundle formation. The spatiotemporal expression of EB2 thus dramatically influences microtubule organisation, EB1 and ACF7 deployment and epithelial differentiation.

A large body of experimental evidence suggests that cytokines trigger pancreatic β-cell death in type 1 diabetes mellitus. Daintain/AIF-1 (Allograft Inflammatory Factor-1), a specific marker for activated macrophages, is accumulated in the pancreatic islets of pre-diabetic BB rats. In the present study, we demonstrate that daintain/AIF-1 is released into blood and the levels of daintain/AIF-1 in the blood of type 1 diabetes-prone non-obese diabetic (NOD) mice suffering from insulitis are significantly higher than that in healthy NOD mice. When injected intravenously into NOD mice, daintain/AIF-1 stimulates white blood cell proliferation, increases the concentrations of blood glucose, impairs insulin expression, up-regulates nitric oxide (NO) production in pancreases and accelerates diabetes in NOD mice, while the antibody against daintain/AIF-1 delays or prevents insulitis in NOD mice. These results imply daintain/AIF-1 triggers type 1 diabetes probably via arousing immune cells activation and induction of NO production in pancreas of NOD mice., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Previous research has suggested that obesity is associated with increased high on-treatment platelet reactivity. We therefore tested platelet reactivity in 251 patients with VerifyNow P2Y12, vasodilator-stimulated phosphoprotein phosphorylation, and light transmission aggregometry with adenosine diphosphate 5 and 20 μM 6 to 24 hours after percutaneous coronary intervention. High on-treatment platelet reactivity was defined as a maximum platelet aggregation ≥46% for light transmission aggregometry with adenosine diphosphate 5 μM or ≥60% for 20 μM, platelet reactivity index ≥50% for vasodilator-stimulated phosphoprotein phosphorylation, and P2Y12 reaction units ≥235 for VerifyNow. The relation between body mass index (BMI) and platelet reactivity values was examined with Spearman coefficients; BMI and high on-treatment platelet reactivity were assessed with Student's t test. Multivariable logistic regression for high on-treatment platelet reactivity was also performed. Average BMI was 30.3 ± 5.9 kg/m² and 44% of patients had a BMI ≥30 kg/m². Overall there was very poor correlation between BMI and on-treatment platelet reactivity, with Spearman coefficients ranging from 0.08 to 0.10. BMI was also not associated with the various definitions of high on-treatment platelet reactivity. Multivariable logistic regressions showed no association between BMI and high on-treatment platelet reactivity. In conclusion, and contrary to previous reports, we found no association whatsoever between BMI and on-treatment platelet reactivity as quantified by a variety of platelet function tests., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Aim: To determine the effect of various SNPs on post-clopidogrel platelet reactivity and clinical outcome., Materials & Methods: Cytochrome 2C19 (CYP2C19) loss-of-function (LOF; *2, *3) and gain-of-function (GOF; *17) allelic variants, together with ABCB1 (3435 C→T and 2677 G→T/A) and paraoxonase-1 (PON-1; 192 Q→R) SNPs were analyzed in 189 patients after elective stent implantation who participated in a randomized, placebo-controlled trial (NCT00638326). Platelet reactivity was determined with light transmission aggregometry and vasodilator stimulated phosphoprotein phosphorylation (VASP-PRI) 12-24 h after 600 mg clopidogrel. High on-treatment platelet reactivity (HTPR) was defined according to the consensus definition (ADP 5 µM >46%; VASP-PRI>50%)., Results: In the case of CYP2C19 genotypes, a gene-dose effect was observed in ADP reactivity with the lowest values in GOF homozygotes and the highest degree in patients carrying two LOF alleles. The odds for HTPR also increased with the number of LOF alleles. There were no significant differences in platelet reactivity according to PON-1 or ABCB1 genotypes. In multivariate analysis, the presence of a CYP2C19 LOF allele turned out to be the independent determinant of HTPR. Although the study was not powered to clinical outcome (not LOF heterozygotes), only patients with two LOF alleles had a significantly higher risk for cardiovascular death, myocardial infarction or unplanned target vessel revascularization at 1 year compared with non-LOF carriers., Conclusion: Genetic variants in CYP2C19 have a gene-dose effect on post-clopidogrel platelet reactivity, with homozygote LOF carriers having the highest risk for HTPR and for adverse ischemic events. Neither ABCB1 nor PON-1 genotypes significantly influenced platelet reactivity or outcome.

Expression of the src-family kinase lymphocyte-specific protein tyrosine kinase (Lck) at the plasma membrane is essential for it to fulfill its pivotal role in signal transduction in T lymphocytes. MAL, an integral membrane protein expressed in specific types of lymphoma, has been shown to play an important role in targeting Lck to the plasma membrane. Here we report that MAL interacts with Inverted Formin2 (INF2), a formin with the atypical property of promoting not only actin polymerization but also its depolymerization. In Jurkat T cells, INF2 colocalizes with MAL at the cell periphery and pericentriolar endosomes and along microtubules. Videomicroscopic analysis revealed that the MAL(+) vesicles transporting Lck to the plasma membrane move along microtubule tracks. Knockdown of INF2 greatly reduced the formation of MAL(+) transport vesicles and the levels of Lck at the plasma membrane and impaired formation of a normal immunologic synapse. The actin polymerization and depolymerization activities of INF2 were both required for efficient Lck targeting. Cdc42 and Rac1, which bind to INF2, regulate Lck transport in both Jurkat and primary human T cells. Thus, INF2 collaborates with MAL in the formation of specific carriers for targeting Lck to the plasma membrane in a process regulated by Cdc42 and Rac1.

The actin cytoskeleton plays an important role in many cellular processes, including cell mortality, mitosis, cytokinesis, intracellular transport, endocytosis and secretion but also is involved in gene transcription. The dynamics of the actin cytoskeleton is controlled by different classes of actin-binding proteins (ABPs) which regulate the polymerization of actin filaments. In this report we used siRNA against cofilin-1 (nonmuscle) to demonstrate the effect of cofilin on the nuclear and cytoplasmic actin pools in CHO AA8 cells after exposition to various concentrations of doxorubicin. The immunofluorescence studies showed doxorubicin dose dependent tendency to formation the multinucleated giant cells, but also the increase of fluorescence intensity of cofilin in nuclei of untransfected cells. Induction of cell death with doxorubicin treatment in untransfected cells revealed both mitotic catastrophe (in both lower and higher doxorubicin doses) and apoptosis (mostly in higher doxorubicin doses), whereas among cofilin-1 down-regulated cells we observed only mitotic catastrophe. The results suggest that cofilin has apoptosis-inducing ability and that mitotic catastrophe is independent from F-actin content in cell nucleus. In this point of view we conclude that different mechanisms of chromatin reorganization are involved in these two processes. Moreover, we suppose that apoptosis and mitotic catastrophe are independent from each other.

Introduction: High on-treatment platelet reactivity (HTPR) after clopidogrel loading dose (LD) is associated with a high risk of thrombotic events after percutaneous coronary intervention(PCI). We have demonstrated that HTPR could be overcome in the majority of cases using LD adjustment resulting in an improved clinical outcome. However this strategy failed in nearly 10% of patients with HTPR. We aimed to determine if P2Y12-ADP receptor polymorphisms were associated with failed dose adjustment., Material and Method: Forty-three patients undergoing PCI were included in this prospective study. A VASP index >or=50% after a 600 mg LD of clopidogrel defined HTPR. Dose adjustment was performed according to PR monitoring to reach a VASP index <50%. Genetic polymorphism of the P2Y12-ADP receptor was determined by direct sequencing., Results: Patients with successful dose-adjustment (SDA) (n=33) and failed (FDA) (n=10) dose-adjustment groups were compared. The 2 groups were similar in terms of cardiovascular risk factors including diabetes mellitus (SDA vs FDA: 42 vs 20%; p=0.3). The prevalence of the H2 allele of the P2Y12-ADP receptor was also similar between the 2 groups (p=0.3). The H2 allele was found in 6 patients which are all included in the SDA group. After the first 600 mg loading dose of clopidogrel, patients carrying at least one H2 allele had similar VASP index compared to those carrying two copies of the wild type allele (H1) (SDA vs FDA: 44.9+/-14.9 vs 43.5+/-10%; p=0.8)., Conclusion: The present study suggests that the H2 allele of the P2Y12-ADP receptor is not involved in clopidogrel failed dose adjustment according to platelet reactivity monitoring., ((c) 2009. Published by Elsevier Ltd.)

Gelsolin and calponin are cytoskeletal and signalling proteins that form a tight 1:1 complex (GCC). We show that calponin within the GCC inhibits the rate of gelsolin mediated nucleation of actin polymerization. The actin-binding function of calponin is ablated within the GCC as the actin-binding site overlaps with one of the gelsolin binding sites. The structure of filaments that result from nucleation by GCC are different to those nucleated by gelsolin alone in that they are longer, loosely bundled and stain heterogeneously with phalloidin. GCC nucleated filaments appear contorted and wrap around each to form the loose bundles., (2009 Elsevier Inc. All rights reserved.)

The messenger-RNA (mRNA) expression of selected cytokines and chemokines in primary chicken oviduct epithelial cells (COEC) was determined following in vitro infections with wild-type or type three secretion system (T3SS)-mutant Salmonella enterica serovar Enteritidis (SE) strains. All SE strains examined in this study elicited the expression of proinflammatory immune mediators including inducible nitric oxide synthase (iNOS), CXCLi1 (K60), CXCLi2 (IL-8), CCLi3 (K203), and CCLi4 (MIP-1beta). SE also triggered the expression of an anti-inflammatory cytokine, IL-10, but repressed TGF-beta3 transcription. Both T3SS-1 (sipA and sipB) and T3SS-2 (pipB and ssaV) mutants showed reduced capacity, compared to the wild-type SE, to stimulate iNOS mRNA expression in COEC. T3SS-1 (sipA and sipB) mutants were significantly impaired in their ability to induce the expression of CXCLi1 and CXCLi2. T3SS-2 mutants displayed a wild-type phenotype in terms of modulating the expression of chemokines and cytokines in COEC. The expression of iNOS, but not CXC chemokines, correlated with the number of intracellular bacteria in COEC. Genetic complementation of the sipA mutation restored a wild-type phenotype. Thus, SE induction of CXCLi1 and CXCLi2 was sipA-dependent. These results provide enhanced insights into the complex interplay between local host innate immune system and bacterial virulence factors.

Elastin is the polymeric protein responsible for the properties of extensibility and elastic recoil of the extracellular matrix in a variety of tissues. Although proper assembly of the elastic matrix is crucial for its durability, the process by which this assembly takes place is not well-understood. Recent data suggest the complex interaction of tropoelastin, the monomeric form of elastin, with a number of other elastic matrix-associated proteins, including fibrillins, fibulins, and matrix-associated glycoprotein (MAGP), is important to achieve the proper architecture of the elastic matrix. At the same time, it is becoming clear that self-assembly properties intrinsic to tropoelastin itself, reflected in a temperature-induced phase separation known as coacervation, are also important in this assembly process. In this study, using a well-characterized elastin-like polypeptide that mimics the self-assembly properties of full-length tropoelastin, the process of self-assembly is deconstructed into "coacervation" and "maturation" stages that can be distinguished kinetically by different parameters. Members of the fibrillin, fibulin, and MAGP families of proteins are shown to profoundly affect both the kinetics of self-assembly and the morphology of the maturing coacervate, restricting the growth of coacervate droplets and, in some cases, causing clustering of droplets into fibrillar structures.

Sustained nigrostriatal dopamine depletion increases the serine/threonine phosphorylation of multiple striatal proteins that play a role in corticostriatal synaptic plasticity, including Thr(286) phosphorylation of calcium/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha). Mechanisms underlying these changes are unclear, but protein phosphatases play a critical role in the acute modulation of striatal protein phosphorylation. Here we show that dopamine depletion for periods ranging from 3 weeks to 10 months significantly reduces the total activity of protein phosphatase (PP) 1, but not of PP2A, in whole lysates of rat striatum, as measured using multiple substrates, including Thr(286)-autophosphorylated CaMKIIalpha. Striatal PP1 activity is partially inhibited by a fragment of the PP1-binding protein neurabin-I, Nb-(146-493), because of the selective inhibition of the PP1gamma(1) isoform. The fraction of PP1 activity that is insensitive to Nb-(146-493) was unaffected by dopamine depletion, demonstrating that dopamine depletion specifically reduces the activity of PP1 isoforms that are sensitive to Nb-(146-493) (i.e. PP1gamma(1)). However, total striatal levels of PP1gamma(1) or any other PP1 isoform were unaffected by dopamine depletion, and our previous studies showed that total levels of the PP1 regulatory/targeting proteins DARPP-32, spinophilin, and neurabin were also unchanged. Rather, co-immunoprecipitation experiments demonstrated that dopamine depletion increases the association of PP1gamma(1) with spinophilin in striatal extracts. In combination, these data demonstrate that striatal dopamine depletion inhibits a specific synaptic phosphatase by increasing PP1gamma(1) interaction with spinophilin, perhaps contributing to hyperphosphorylation of synaptic proteins and disruptions of synaptic plasticity and/or dendritic morphology.

Autotaxin (ATX) is a potent tumor cell motogen that can produce lysophosphatidic acid (LPA) from lysophosphatidylcholine. LPA is a lipid mediator that has also been shown to modulate tumor cell invasion. Autotaxin mRNA is expressed at significant levels in the intestine. Likewise, LPA2 receptor levels have been shown to be elevated in colon cancers. The molecular mechanism of ATX/LPA-induced increase in intestinal cell migration however, remains poorly understood. Villin is an intestinal and renal epithelial cell specific actin regulatory protein that modifies epithelial cell migration. In this study we demonstrate that both Caco-2 (endogenous villin) and MDCK (exogenous villin) cells, which express primarily LPA2 receptors, show enhanced cell migration in response to ATX/LPA. ATX and LPA treatment results in the rapid formation of lamellipodia and redistribution of villin to these cell surface structures, suggesting a role for villin in regulating this initial event of cell locomotion. The LPA-induced increase in cell migration required activation of c-src kinase and downstream tyrosine phosphorylation of villin by c-src kinase. LPA stimulated cell motility was determined to be insensitive to pertussis toxin, but was regulated by activation of PLC-gamma 1. Together, our results show that in epithelial cells ATX and LPA act as strong stimulators of cell migration by recruiting PLC-gamma 1 and villin, both of which participate in the initiation of protrusion.

Prostate tumors are initially dependent on androgens for growth, but the majority of patients treated with anti-androgen therapy progress to androgen-independence characterized by resistance to such treatment. This study investigates a novel role for filamin A (FlnA), a 280 kDa cytoskeletal protein (consisting of an actin-binding domain (ABD) followed by 24 sequential repeats), in androgen-independent (AI) growth. Full-length FlnA is cleaved to 170 kDa (ABD+FlnA1-15) and 110 kDa fragments (FlnA16-24); the latter is further cleaved to a 90 kDa fragment (repeats 16-23) capable of nuclear translocation and androgen receptor (AR) binding. Here, we demonstrate that in androgen-dependent LNCaP prostate cancer cells, the cleaved 90 kDa fragment is localized to the nucleus, whereas in its AI subline C4-2, FlnA failed to cleave and remained cytoplasmic. Transfection of FlnA16-24 cDNA in C4-2 cells restored expression and nuclear localization of 90 kDa FlnA. Unlike LNCaP, C4-2 cells proliferate in androgen-reduced medium and in the presence of the AR-antagonist Casodex. They also exhibit increased Akt phosphorylation compared to LNCaP, which may contribute to their AI phenotype. Nuclear expression of 90 kDa FlnA in C4-2 cells decreased Akt phosphorylation, prevented proliferation in androgen-reduced medium and restored Casodex sensitivity. This effect was inhibited by constitutive activation of Akt indicating that FlnA restored Casodex sensitivity in C4-2 cells by decreasing Akt phosphorylation. In addition, FlnA-specific siRNA which depleted FlnA levels, but not control siRNA, induced resistance to Casodex in LNCaP cells. Our results demonstrate that expression of nuclear FlnA is necessary for androgen dependence in these cells.

One-trial conditioning in Hermissenda results in enhanced intrinsic cellular excitability of sensory neurons in the conditioned stimulus pathway, and the phosphorylation of several proteins. Previous results demonstrated that the development of enhanced intrinsic excitability was dependent on the expression of conditioned stimulus pathway phosphoprotein-24 (Csp24), an intracellular protein containing four repeated beta-thymosin homology domains. Consistent with this, antisense oligonucleotide-mediated inhibition of Csp24 expression prevents the reduction in amplitude of the A-type transient K+ current (I(A)) and the depolarized shift in the steady-state activation curve normally produced by one-trial in vitro conditioning of isolated photoreceptors. One-trial conditioning also regulates Csp24 phosphorylation. We now show that purified recombinant Csp24 sequesters G-actin in vitro with an approximate K(d) value of 2.8 microM. We also observed a significant increase in the coprecipitation of actin with Csp24 after one-trial in vitro conditioning using antibodies directed toward either Csp24 or phospho-Csp24. Preincubation with protein kinase C (PKC) selective inhibitors attenuated the increase in Csp24 phosphorylation and coprecipitated actin observed after one-trial conditioning. Our findings indicate that the PKC signaling pathway contributes to the phosphorylation of Csp24 after one-trial conditioning, and that PKC activity modulates an association between Csp24 and actin. These data suggest Csp24 may influence intrinsic excitability by regulating cytoskeletal dynamics.

Neurabin is a brain-specific actin and protein phosphatase-1 (PP-1) binding protein that inhibits the purified catalytic subunit of protein phosphatase-1 (PP-1(C)). However, endogenous PP-1 exists primarily as multimeric complexes of PP-1(C) bound to various regulatory proteins that determine its activity, substrate specificity, subcellular localization and function. The major form of endogenous PP-1 in brain is protein phosphatase-1(I) (PP-1(I)), a Mg(2+)/ATP-dependent form of PP-1 that consists of PP-1(C), the inhibitor-2 regulatory subunit, an activating protein kinase and other unidentified proteins. We have identified four PP-1(I) holoenzyme fractions (PP-1(IA), PP-1(IB), PP-1(IC), and PP-1(ID)) in freshly harvested pig brain separable by poly-L-lysine chromatography. Purified recombinant neurabin (amino acid residues 1-485) inhibited PP-1(IB) (IC(50)=1.1 microM), PP-1(IC) (IC(50)=0.1 microM), and PP-1(ID) (IC(50)=0.2 microM), but activated PP-1(IA) by up to threefold (EC(50)=40 nM). The PP-1(IA) activation domain was localized to neurabin(1-210). Our results indicate a novel mechanism of PP-1 regulation by neurabin as both an inhibitor and an activator of distinct forms of PP-1(I) in brain.

The purpose of this study was to determine the effect of thymosin beta 4 (Tbeta4) on NFkappaB protein levels, activation, phosphorylation, and nuclear translocation in a model of tumor necrosis factor (TNF)-alpha-mediated corneal inflammation. Transformed and primary (HCET and HCEC) human corneal epithelial cells were stimulated with the pro-inflammatory cytokine TNF-alpha and treated or not with Tbeta4. Nuclear NFkappaB p65 subunit protein levels were assayed using ELISA, and activity was measured by determining NFkappaB binding to consensus oligonucleotides. NFkappaB p65 protein phosphorylation was also measured by ELISA. Nuclear translocation of NFkappaB p65 subunit was assayed by immunofluorescence microscopy. Compared to non-treated controls, Tbeta4 treatment significantly decreased nuclear NFkappaB protein levels, NFkappaB activity and p65 subunit phosphorylation in corneal epithelial cells after TNF-alpha stimulation. In TNF-alpha-stimulated corneal epithelial cells, NFkappaB p65 subunit translocation to the nucleus was observed using immunofluorescence microscopy. In contrast, Tbeta4 blocked nuclear translocation of the NFkappaB p65 subunit in TNF-alpha-stimulated corneal epithelial cells. TNF-alpha initiates cell signaling pathways that converge on the activation of NFkappaB, thus both are known mediators of the inflammatory process. Tbeta4, a protein with diverse cellular functions including wound healing and suppression of inflammation, inhibits the activation of NFkappaB in TNF-alpha-stimulated cells. These results have important clinical implications for the potential role of Tbeta4 as a corneal anti-inflammatory agent.

Arrays of >5,000 Saccharomyces cerevisiae proteins were screened to identify proteins that can preferentially bind a small RNA hairpin that contains a clamped adenine motif (CAM). A CAM is required for the replication of Brome Mosaic Virus (BMV), a plant-infecting RNA virus that can replicate in S. cerevisiae. Several hits were selected for further characterization in Nicotiana benthamiana. Pseudouridine Synthase 4 (Pus4) and the Actin Patch Protein 1 (App1) modestly reduced BMV genomic plus-strand RNA accumulation, but dramatically inhibited BMV systemic spread in plants. Pus4 also prevented the encapsidation of a BMV RNA in plants and the reassembly of BMV virions in vitro. These results demonstrate the feasibility of using proteome arrays to identify specific RNA-binding proteins for antiviral activities. Furthermore, the effects of Pus4 suggest that the CAM-containing RNA motif provides a regulatory link between RNA replication and encapsidation.

Background: The primary cause of early death in untreated Marfan syndrome (MFS) patients is aortic dilatation and dissection., Methods and Results: We investigated whether ascending aortic samples from the fibrillin-1-underexpressing mgR mouse model for MFS or a recombinant fibrillin-1 fragment containing an elastin-binding protein (EBP) recognition sequence can act as chemotactic stimuli for macrophages. Both the aortic extracts from the mgR/mgR mice and the fibrillin-1 fragment significantly increased macrophage chemotaxis compared with extracts from wild-type mice or buffer controls. The chemotactic response was significantly diminished by pretreatment of macrophages with lactose or with the elastin-derived peptide VGVAPG and by pretreatment of samples with a monoclonal antibody directed against an EBP recognition sequence. Mutation of the EBP recognition sequence in the fibrillin-1 fragment also abolished the chemotactic response. These results indicate the involvement of EBP in mediating the effects. Additionally, investigation of macrophages in aortic specimens of MFS patients demonstrated macrophage infiltration in the tunica media., Conclusions: Our findings demonstrate that aortic extracts from mgR/mgR mice can stimulate macrophage chemotaxis by interaction with EBP and show that a fibrillin-1 fragment possesses chemotactic stimulatory activity similar to that of elastin degradation peptides. They provide a plausible molecular mechanism for the inflammatory infiltrates observed in the mgR mouse model and suggest that inflammation may represent a component of the complex pathogenesis of MFS.

Protein phosphatase-1 (PP1) has been implicated in the control of long-term potentiation (LTP) and depression (LTD) in rat hippocampal CA1 neurons. PP1 catalytic subunits associate with multiple postsynaptic regulatory subunits, but the PP1 complexes that control hippocampal LTP and LTD in the rat hippocampus remain unidentified. The neuron-specific actin-binding protein, neurabin-I, is enriched in dendritic spines, and tethers PP1 to actin-rich postsynaptic density to regulate morphology and maturation of spines. The present studies utilized Sindbis virus-mediated expression of wild-type and mutant neurabin-I polypeptides in organotypic cultures of rat hippocampal slices to investigate their role in synaptic plasticity. While wild-type neurabin-I elicited no change in basal synaptic transmission, it enhanced LTD and inhibited LTP in CA1 pyramidal neurons. By comparison, mutant neurabins, specifically those unable to bind PP1 or F-actin, decreased basal synaptic transmission, attenuated LTD and increased LTP in slice cultures. Biochemical and cell biological analyses suggested that, by mislocalizing synaptic PP1, the mutant neurabins impaired the functions of endogenous neurabin-PP1 complexes and modulated LTP and LTD. Together, these studies provided the first biochemical and physiological evidence that a postsynaptic actin-bound neurabin-I-PP1 complex regulates synaptic transmission and bidirectional changes in hippocampal plasticity.

Photo-switchable compounds are becoming increasingly popular for a series of biological applications based on the reversible photo-control of structure and function of biomolecules. Three applications for the usage of BODTCM and hemithioindigo as photo-reactive compounds are described here. The structure of the villin headpiece was modified by replacing a part of the backbone with hemithioindigo, aiming at induction of the folding process by irradiation with a defined wavelength. The E-isomer of BODTCM was applied as potential inhibitor of the 12/15-lipoxygenase (12/15-LOX), which is implicated in the pathogenesis of inflammatory diseases. A required death domain for the binding of proapoptotic proteins (e.g. Bak) to the hydrophobic groove of antiapoptotic proteins is the BH3 helix. Inserting hemithioindigo into this short peptide, stabilization towards proteolytic degradation is achieved. Such photo-reactive compounds might be developed as potential drugs for a great variety of diseases.

In addition to mediating cell signalling events, native extracellular matrix (ECM) assemblies interact with other ECM components, act as reservoirs for soluble signalling molecules and perform structural roles. The potential of native ECM assemblies in the manufacture of biomimetic materials has not been fully exploited due, in part, to the effects of substrate interactions on their morphology. We have previously demonstrated that the ECM components, fibrillin and type VI collagen microfibrils, exhibit substrate dependent morphologies on chemically and topographically variable heterogeneous surfaces. Using both cleaning and coating approaches on silicon wafers and glass coverslips we have produced chemically homogeneous, topographically similar substrates which cover a large amphiphilic range. Extremes of substrate amphiphilicity induced morphological changes in periodicity, curvature and lateral spreading which may mask binding sites or disrupt domain structure. Biological functionality, as assayed by the ability to support cell spreading, was significantly reduced for fibrillin microfibrils adsorbed on highly hydrophilic substrates (contact angle 20.7 degrees) compared with less hydrophilic (contact angle 38.3 degrees) and hydrophobic (contact angle 92.8 degrees) substrates. With an appropriate choice of surface chemistry, multifunctional ECM assemblies retain their native morphology and biological functionality.

To explore the possible role of the thin filament-linked regulation of cross-bridge cycling in living smooth muscle contraction, we studied the effects of TnIp and HSP20p, a synthetic peptide originating from an actin tropomyosin binding region of rabbit cardiac troponin I (residues 136-147; GKFKRPTLRRVR), and that of human heat shock protein 20 (residues 110-121; GFVAREFHRRYR) on the relaxation of skinned (cell membrane ilized) preparations from guinea pig taenia caeci. An active stress of the skinned preparations, resulting from actin-myosin interaction, rapidly decayed following Ca(2+) removal (relaxation). TnIp accelerated the initial rapid phase and slowed the following slow phase of the relaxation. On the other hand, HSP20p only slowed the whole process of the relaxation. The relaxation time courses were well fitted in a double exponential manner, and the double exponential decay of the stress could be explained as a portion of fast-detaching cross bridges not to dissociate rapidly by Ca(2+) removal, but to transfer to latch bridges dissociating very slowly. Our present results suggested that (i) TnIp and HSP20p accelerated transferring from fast-detaching cross bridges to slow-detaching (latch) bridges, and (ii) TnIp accelerated dissociation of the fast-detaching cross bridges and the latch bridges, while HSP20p slowed dissociation the fast-detaching cross bridges. Since TnIp and HSP20p are thought to bind to actin and tropomyosin, but not to myosin, we concluded that through thin-filament-dependent mechanisms these peptides regulated the formation and/or deformation of latch bridges in smooth muscle. The thin-filament-dependent regulation might physiologically control the stress maintenance and relaxation in smooth muscle cells.

The neuronal nuclear matrix protein, NRP/B, contains a BTB domain and kelch repeats and is expressed in primary neurons but not in primary glial cells. To examine the function of NRP/B in neurons, we analyzed the structure/function of the NRP/B-BTB domain and its role in neurite outgrowth. Based on three-dimensional modeling of NRP/B, we generated an NRP/B-BTB mutant containing three mutations in the conserved amino acids D47A, H60A and R61D that was termed BTB mutant A. BTB mutant A significantly reduced the dimerization of NRP/B compared to wild-type NRP/B. The NRP/B-BTB domain was required for nuclear localization and mediated the association of NRP/B with p110RB through the TR subdomain within the B pocket of p110RB. Overexpression of wild-type NRP/B and NRP/B-BTB domain significantly induced neurite outgrowth in PC12 cells and enhanced the G0-G1 cell population by approximately 23% compared to the control cells, whereas NRP/B-BTB mutant A reduced neurite outgrowth by 70-80%, and inhibited NRP/B-p110RB association. Single cell microinjection of NRP/B-specific antibodies also blocked the neurite outgrowth of PC12 cells upon NGF stimulation. Interference of NRP/B expression by small interfering RNA (NRP/B-siRNA) inhibited neurite outgrowth and suppressed the NGF-induced outgrowth of neurites in PC12 cells. Additionally, p110RB phosphorylation at serine residue 795 was significantly reduced in PC12 cells treated with NRP/B siRNA compared to those treated with control GFP-siRNA, indicating that p110RB is a downstream target of NRP/B. Thus, the BTB domain of NRP/B regulates neurite outgrowth through its interaction with the TR subdomain within the B pocket of p110RB, and the conserved amino acids D47A, H60A and R61D within this domain of NRP/B are crucial residues for neurite extension in neuronal cells. These findings support a role for the BTB-domain of NRP/B as an important regulator of neuronal differentiation.

Rearrangement of the cytoskeleton leading to the extension of cellular processes is essential for the myelination of axons by oligodendrocytes. We observed that the actin-binding protein, Mayven, is expressed during all stages of the oligodendrocyte lineage, and that its expression is up-regulated during oligodendrocyte differentiation. Mayven is localized in the cytoplasm and along the cell processes. Mayven also binds actin, and is involved in the cytoskeletal reorganization in oligodendrocyte precursor cells (O-2A cells) that leads to process elongation. Mayven overexpression resulted in an increase in the process outgrowth of O-2A cells and in the lengths of the processes, while microinjection of Mayven-specific antibodies inhibited process extension in these cells. Furthermore, O-2A cells transduced with recombinant retroviral sense Mayven (pMIG-W-Mayven) showed an increase in the number of oligodendrocyte processes with outgrowth, while recombinant retroviral antisense Mayven (pMIG-W-Mayven-AS) blocked O-2A process extension. Interestingly, co-localization and association of Mayven with Fyn kinase were found in O-2A cells, and these interactions were increased during the outgrowth of oligodendrocyte processes. This association was mediated via the SH3 domain ligand (a.a. 1-45) of Mayven and the SH3 domain of Fyn, suggesting that Mayven may act as a linker to bind Fyn, via its N-terminus. Thus, Mayven plays a role in the dynamics of cytoskeletal rearrangement leading to the process extension of oligodendrocytes.

CDT from Clostridium difficile is an ADP-ribosyltransferase that causes rapid actin disaggregation and cell death. For efficient catalysis, CDT required specific divalent cations and binding by NAD which can be substituted by ATP but not ADP. Increasing isolation of CDT-producing strains prompted our search for antagonists like the anti-C. difficile agents bacitracin and vancomycin which were effective CDT inhibitors. Other CDT transferase and glycohydrolase inhibitors with consistently low IC50 values were heterocyclic peptide antibiotics containing modified amino acids such as polymyxin B and beta-lactam cephalosporins. The strongest inhibitors were actin-binding proteins which possess extensive interfaces with G-actin, adjoining the CDT-ADP-ribose+ acceptor site and nucleotide cleft. Analysis of the extent and mode of inhibition and actin interaction sites provided fresh evidences on the designation of actin interface domains with actin-binding proteins. Our results uphold ADP-ribosylation as an innate physiologic process in cellular cytoskeletal reorganization regulated by endogenous metabolites.

Overexpression of androgen receptors (AR) in PC-3 cell, and treatment of 5alpha-dihydrotestosterone in LNCaP cells lead to the suppression of VDR transactivation. Competition for shared coregulators between AR and VDR is one possible mechanism to explain the suppressive effect of androgen-AR signals on VDR activity. Among the AR coregulators we tested, ARA54, ARA70, supervillin, and gelsolin were found to enhance VDR transactivation. Further characterization of the interaction between ARA54 or ARA70 and VDR demonstrated a direct interaction between VDR and ARA70, but no association between ARA54 and VDR. The LXXLL motif of ARA70 is essential for interaction with VDR and partially responsible for its function as a coactivator of VDR. The suppression of VDR transactivation by AR signal was restored by overexpression of ARA70, but not ARA54. Together, ARA70 and ARA54 modulate VDR transactivation, and the competition for ARA70 mediates the suppressive effect of androgen-AR on VDR transactivation.

Fesselin is an actin binding protein that bundles actin filaments and accelerates nucleation of actin polymerization. The effect of fesselin on actin polymerization is regulated by Ca(++)-calmodulin. Because actin filaments serve both structural and contractile functions we also examined the effect of fesselin on activation of myosin S1 ATPase activity. Fesselin inhibited the activation of S1-catalyzed ATP hydrolysis in a similar manner in both the presence and absence of tropomyosin. This inhibition was unaffected by Ca(++)-calmodulin. Fesselin inhibited the binding of myosin-S1 to actin during steady-state ATP hydrolysis. Fesselin also displaced caldesmon from actin. S1 displaced fesselin from actin in the absence of nucleotide when the affinity of S1 for actin was much greater than the affinity of fesselin for actin. It is likely that fesselin and S1 share common binding sites on F-actin. We also observed that fesselin could bind to smooth muscle myosin with muM affinity. Fesselin shares some similarities to caldesmon in binding to several other proteins and having multiple potential functions.

Actin, one of the most highly conserved and abundant eukaryotic proteins, is constantly being polymerized and depolymerized within cells as part of cellular motility, tissue formation and repair, and embryonic development. Many proteins exist that bind to monomeric or filamentous (F) forms of actin to regulate the polymerization state. It has become increasingly apparent that the ability of different proteins to bind to and regulate actin filament dynamics depends on the ability of the filament to exist in altered conformations. Yet, little is known about how these conformational changes occur at the molecular level. We have destabilized F-actin filaments by forming a disulfide that locks the "hydrophobic plug" to the body of the actin subunit or by altering the C terminus of actin with a tetramethylrhodamine label. We also examined F-actin filaments at short times after the initiation of polymerization. In all three cases, a substantial fraction of protomers can be found in a "tilted" state that also is induced by actin depolymerizing factor/cofilin proteins. These observations suggest that F-actin filaments are annealed over time into a stable filament and that actin-depolymerizing proteins can effect a time reversal of polymerization.