Yes-associated protein ameliorates tumor necrosis factor-α-induced epithelial barrier dysfunction by affecting the F-actin / 中国小儿急救医学

Objective:To investigate the protective role of Yes-associated protein(YAP)in intestinal epithelial barrier injury.Methods:The intestinal epithelial barrier model was established by culturing human colorectal adenocarcinoma cell line Caco-2 cells, which were divided into four groups: control group, Caco-2 monolayers did not receive any treatment; recombinant human tumor necrosis factor-α(rhTNF-α)group, 100 μg/L of rhTNF-α was added to Caco-2 monolayers; vector+ rhTNF-α group, Caco-2 monolayers were first added with control plasmid pcDNA3.1-vector, and 100 μg/L rhTNF-α was added 24 hours later; YAP+ rhTNF-α group, Caco-2 cells with barrier construction were first added with pcDNA3.1-YAP, and 100 μg/L rhTNF-α was added 24 hours later.Realtime-PCR and Western blot were used to evaluate YAP mRNA and protein expression level.Epithelial permeability was assayed by trans-epithelial electrical resistance(TEER)and fluorescein isothiocyanate-dextran 40(FD-40 flu). Cellular distribution of F-actin was assayed by immunofluorescence staining.Results:Compared with control group[(607.3±29.3)Ω·cm 2], TEER of rhTNF-α group[(265.3±32.7)Ω·cm 2] decreased, while TEER of YAP+ rhTNF-α group[(387.0±18.7)Ω·cm 2]increased compared with rhTNF-α group, the differences were statistically significant( P<0.001). The FD-40 flux of rhTNF-α(22.7%±0.5%) group was higher than that of the control group(6.3%±0.9%), while the FD-40 flux of Yap + rhTNF-α group(12.2%±0.8%) was lower than that of rhTNF-α group, the differences were statistically significant( P<0.001). Immunofluorescence staining showed that compared with the control group, the cytoskeletal F-actin fiber dense spot decreased in rhTNF-α group, and some cells showed obvious trans-cellular stress fiber structure, while the peripheral actin band was clear in YAP+ rhTNF-α group, and the intracellular stress fiber decreased.YAP+ TNF-α group appeared as a clear, peripheral actin ribbon with a decrease in cytoplasmic stress fibres. Conclusion:YAP overexpression significantly inhibits TNF-α induced decline of TEER, and increases of FD-40 flux and F-actin rearrangement of Caco-2.YAP could ameliorate TNF-α induced intestinal epithelial barrier injury by regulating cytoskeleton F-actin.

Ultrastructural clarification of the peripherally located actin network in the myelinated axons / Aclaración ultraestructural de la red de actina localizada periféricamente en los axones mielinizados

SUMMARY: Despite the existence of a large amount of actin in the axons, the concentration F-actin was quite low in the myelinated axons and almost all the F-actin were located in the peripheries of the myelinated axons. Until now, the ultrastructural localization of F-actin has still not been reported in the myelinated axons, probably due to the lack of an appropriate detection method. In the present study, a phalloidin-based FITC-anti-FITC technique was adopted to investigate the subcellular localization of F-actin in the myelinated axons. By using this technique, F-actin is located in the outer and inner collars of myelinated cytoplasm surrounding the intermodal axon, the Schmidt-Lanterman incisures, the paranodal terminal loops and the nodal microvilli. In addition, the satellite cell envelope, which encapsulates the axonal initial segment of the peripheral sensory neuron, was also demonstrated as an F-actin-enriched structure. This study provided a hitherto unreported ultrastructural view of the F-actin in the myelinated axons, which may assist in understanding the unique organization of axonal actin cytoskeleton.

RESUMEN: A pesar de la existencia de una gran cantidad de actina en los axones, la concentración de F-actina era bastante baja en los axones mielinizados y casi la totalidad de F-actina se localizaba en las periferias de los axones mielinizados. A la fecha aún no se ha reportado la localización ultraestructural de F-actina en los axones mielinizados, probablemente debido a la falta de un método de detección apropiado. En el presente estudio, se adoptó una técnica FITC-anti-FITC basada en faloidina para investigar la localización subcelular de F-actina en los axones mielinizados. Mediante el uso de esta técnica, la F-actina se localiza en los collares externo e interno del citoplasma mielinizado que rodea el axón intermodal, a las incisiones de Schmidt-Lanterman,a las asas terminales paranodales y a las microvellosidades nodales. Además, la envoltura de la célula satélite, que encapsula el segmento axonal inicial de la neurona sensorial periférica, también se demostró como una estructura enriquecida con F-actina. Este estudio proporcionó una vista ultraestructural de la F-actina en los axones mielinizados, que puede ayudar a comprender la organización única del citoesqueleto de actina axonal.

Main components in butyl alcohol extract of Baitouweng Decoction inhibited neutrophil chemotaxis / 中国中药杂志

The present study aims to investigate the effects of the main components(aesculin, berberine hydrochloride, and anemoside B4) in the butyl alcohol extract of Baitouweng Decoction(BAEB) on the chemotaxis of neutrophils induced by dimethyl sulfoxide(DMSO). HL60 cells were cultivated in RPMI-1640 complete medium, and transferred into a 6-well plate(2 × 10~5 per mL) with 4 mL in each well, followed by incubation with DMSO at 1.3% for five days. The morphologic changes of cells were observed under an inverted microscope. The CD11 b expression after DMSO induction was analyzed by flow cytometry. The effects of aesculin, berberine hydrochloride, and anemoside B4 on the cell proliferation and migration were detected by CCK8 assay and Transwell assay, respectively. The effects of the main components on the production and polarization of F-actin protein were also examined by flow cytometry and laser confocal microscopy. PI3 K/Akt signaling pathway was checked by Western blot. As revealed by the results, neutrophil-like HL60 cells were observed after DMSO induction. The CD11 b expression in these cells increased significantly as indicated by the flow cytometry. Additionally, 100 μg·mL~(-1) aesculin, 8 μg·mL~(-1) berberine hydrochloride, and 80 μg·mL~(-1) anemoside B4 were potent in inhibiting the migration of neutrophils and reducing F-actin expression. Berberine hydrochloride was verified to be capable of diminishing phosphorylated PI3 K/Akt protein expression. The findings indicate that aesculin, anemoside B4, and especially berberine hydrochloride in the BAEB can inhibit the chemotaxis of neutrophils, which is possibly achieved by the inhibition of F-actin and PI3 K/Akt signaling pathway.

TMEM16A inhibits angiotensin II-induced basilar artery smooth muscle cell migration in a WNK1-dependent manner

Vascular smooth muscle cell (VSMC) migration plays a critical role in the pathogenesis of many cardiovascular diseases. We recently showed that TMEM16A is involved in hypertension-induced cerebrovascular remodeling. However, it is unclear whether this effect is related to the regulation of VSMC migration. Here, we investigated whether and how TMEM16A contributes to migration in basilar artery smooth muscle cells (BASMCs). We observed that AngII increased the migration of cultured BASMCs, which was markedly inhibited by overexpression of TMEM16A. TMEM16A overexpression inhibited AngII-induced RhoA/ROCK2 activation, and myosin light chain phosphatase (MLCP) and myosin light chain (MLC20) phosphorylation. But AngII-induced myosin light chain kinase (MLCK) activation was not affected by TMEM16A. Furthermore, a suppressed activation of integrin

NC1-peptide derived from collagen α3 (IV) chain is a blood-tissue barrier regulator: lesson from the testis / 亚洲男科学杂志(英文版)

Collagen α3 (IV) chains are one of the major constituent components of the basement membrane in the mammalian testis. Studies have shown that biologically active fragments, such as noncollagenase domain (NC1)-peptide, can be released from the C-terminal region of collagen α3 (IV) chains, possibly through the proteolytic action of metalloproteinase 9 (MMP9). NC1-peptide was shown to promote blood-testis barrier (BTB) remodeling and fully developed spermatid (e.g., sperm) release from the seminiferous epithelium because this bioactive peptide was capable of perturbing the organization of both actin- and microtubule (MT)-based cytoskeletons at the Sertoli cell-cell and also Sertoli-spermatid interface, the ultrastructure known as the basal ectoplasmic specialization (ES) and apical ES, respectively. More importantly, recent studies have shown that this NC1-peptide-induced effects on cytoskeletal organization in the testis are mediated through an activation of mammalian target of rapamycin complex 1/ribosomal protein S6/transforming retrovirus Akt1/2 protein (mTORC1/rpS6/Akt1/2) signaling cascade, involving an activation of cell division control protein 42 homolog (Cdc42) GTPase, but not Ras homolog family member A GTPase (RhoA), and the participation of end-binding protein 1 (EB1), a microtubule plus (+) end tracking protein (+TIP), downstream. Herein, we critically evaluate these findings, providing a critical discussion by which the basement membrane modulates spermatogenesis through one of its locally generated regulatory peptides in the testis.

Phytoglycoprotein isolated from Dioscorea batatas Decne promotes intestinal epithelial wound healing / 中国天然药物

Dioscorea batatas Decne (DBD) has been used to heal various illnesses of the kidney and intestine as an herbal medicine in Asia. As a source of therapeutic agents, many glycoproteins have been isolated from mushrooms and plants, but the functional role of glycoprotein in intestinal epithelial wound healing has not been understood yet. In the present study, we investigated the wound healing potentials of the 30 kDa glycoprotein (DBD glycoprotein) isolated from DBD in human intestinal epithelial (INT-407) cells. We found that DBD glycoprotein (100 μg·mL) significantly increased the motility of INT-407 cells for 24 h by activating protein kinase C (PKC). DBD glycoprotein stimulated the activation of p38 mitogen-activated protein kinase (MAPK), which is responsible for the phosphorylation of NF-κB inhibitor α (IκBα). DBD glycoprotein increased the level of profilin-1 (PFN1), α-actinin and F-actin expression via activation of transcription factor, nuclear factor-kappa B (NF-κB) during its promotion of cell migration. Experimental mouse colitis was induced by adding dextran sulfate sodium (DSS) to the drinking water at a concentration of 4% (W/V) for 7 days. We figured out that administration of DBD glycoprotein (10 and 20 mg·kg) lowers the levels of disease activity index and histological inflammation in DSS-treated ICR mice. In this regard, we suggest that DBD glycoprotein has ability to promote the F-actin-related migration signaling events via activation of PKC and NF-κB in intestinal epithelial cells and prevent inflammatory bowel disease.

Spreading Shape and Area Regulate the Osteogenesis of Mesenchymal Stem Cells

BACKGROUND: Mesenchymal stem cells (MSCs) have strong self-renewal ability and multiple differentiation potential. Some studies confirmed that spreading shape and area of single MSCs influence cell differentiation, but few studies focused on the effect of the circularity of cell shape on the osteogenic differentiation of MSCs with a confined area during osteogenic process.METHODS: In the present study, MSCs were seeded on a micropatterned island with a spreading area lower than that of a freely spreading area. The patterns had circularities of 1.0 or 0.4, respectively, and areas of 314, 628, or 1256 µm² . After the cells were grown on a micropatterned surface for 1 or 3 days, cell apoptosis and F-actin were stained and analyzed. In addition, the expression of β-catenin and three osteogenic differentiation markers were immunofluorescently stained and analyzed, respectively.RESULTS: Of these MSCs, the ones with star-like shapes and large areas promoted the expression of osteogenic differentiation markers and the survival of cells. The expression of F-actin and its cytosolic distribution or orientation also correlated with the spreading shape and area. When actin polymerization was inhibited by cytochalasin D, the shape-regulated differentiation and apoptosis of MSCs with the confined spreading area were abolished.CONCLUSION: This study demonstrated that a spreading shape of low circularity and a larger spreading area are beneficial to the survival and osteogenic differentiation of individual MSCs, which may be regulated through the cytosolic expression and distribution of F-actin.

Regulation of blood-testis barrier dynamics by the mTORC1/rpS6 signaling complex: An in vitro study / 亚洲男科学杂志(英文版)

During spermatogenesis, developing germ cells that lack the cellular ultrastructures of filopodia and lamellipodia generally found in migrating cells, such as macrophages and fibroblasts, rely on Sertoli cells to support their transport across the seminiferous epithelium. These include the transport of preleptotene spermatocytes across the blood-testis barrier (BTB), but also the transport of germ cells, in particular developing haploid spermatids, across the seminiferous epithelium, that is to and away from the tubule lumen, depending on the stages of the epithelial cycle. On the other hand, cell junctions at the Sertoli cell-cell and Sertoli-germ cell interface also undergo rapid remodeling, involving disassembly and reassembly of cell junctions, which, in turn, are supported by actin- and microtubule-based cytoskeletal remodeling. Interestingly, the underlying mechanism(s) and the involving biomolecule(s) that regulate or support cytoskeletal remodeling remain largely unknown. Herein, we used an in vitro model of primary Sertoli cell cultures that mimicked the Sertoli BTB in vivo overexpressed with the ribosomal protein S6 (rpS6, the downstream signaling protein of mammalian target of rapamycin complex 1 [mTORC1]) cloned into the mammalian expression vector pCI-neo, namely, quadruple phosphomimetic and constitutively active mutant of rpS6 (pCI-neo/p-rpS6-MT) versus pCI-neo/rpS6-WT (wild-type) and empty vector (pCI-neo/Ctrl) for studies. These findings provide compelling evidence that the mTORC1/rpS6 signal pathway exerted its effects to promote Sertoli cell BTB remodeling. This was mediated through changes in the organization of actin- and microtubule-based cytoskeletons, involving changes in the distribution and/or spatial expression of actin- and microtubule-regulatory proteins.

Effects of n-butylphthalide on mitochondria in hippocampus and learning and memory abilities in rats with chronic alcoholism / 中华行为医学与脑科学杂志

Objective@#To explore the effects of n-butylphthalide (NBP) on mitochondria in hippocampus and learning and memory abilities in rats with chronic alcoholism.@*Methods@#60 male SD rats were randomly divided into three groups on average, including normal group, model group and treatment group, with 20 rats in each group.Rats of model group and treatment group are given 6% (V/V) alcohol solution continuously for 28 d to establish the model of chronic alcoholism.Rats in the treatment group were given butylphthalide for 14 days from the fourteenth day after giving alcohol solution.The Y type electric maze was used to test the learning and memory ability of rats, the content of H2S in the hippocampus and the activity of mitochondrial ATP enzyme were measured by spectrophotometry, and the protein expression of F-actin was detected by Western blot.@*Results@#Compared with the normal group, the learning and memory ability of the rats in the model group were decreased, the content of H2S in the hippocampus were increased, and the activity of mitochondrial ATP enzyme and the expression of F-actin protein were decreased, and most of the mitochondria were damaged under the electron microscope.The training times of the rats in treatment group(61.88±3.61)was lower than that of the model group(82.19±4.87), the ability of learning and memory was improved(P<0.05). Compared with the model group ((1.50±0.07)U/mgprot, (0.08±0.01)), the activity of the mitochondrial ATP enzyme((1.84±0.11)U/mgprot) and the level of F-actin protein(0.12±0.01)in rat hippocampus of treatment group were increased, the difference was statistically significant(both P<0.05). The level of H2S in rat hippocampus of the treatment group ((34.56±2.47) nmol/g) was lower than that of the model group ((44.55±3.71) nmol/g), the difference was statistically significant(P<0.05). Compared with model group, the mitochondrial damage of the hippocampus in the treatment group was improved under electron microscope.@*Conclusion@#NBP can abate mitochondrial damage and improve learning and memory abilities in chronic alcoholism rats.

Role of cell adhesion molecule L1 like in the inhibition of the metastasis of esophageal squamous cell carcinoma / 中华消化杂志

Objective To investigate the role of cell adhesion molecule L1 like (CALL) in the genesis and development of esophageal squamous cell carcinoma (ESCC).Methods From July 2007 to December 2010,a total of 100 patients with ESCC who received radical resection of esophageal cancer were enrolled.The ESCC tissues and corresponding tumor-adjacent normal tissues were obtained.The expression of CALl was determined by tissue microarray technology and immunohistochemical staining.The CALL over-expressed esophageal cancer cell line was established.The effects of CALL on cell migration and invasion were detected by wound-healing assay and Transwell assay,respectively.The effects of CALL on actin microfilament was analyzed by filamentous actin (F-actin) staining.Chi square test,Fisher's exact test,multivariate analysis and t test were performed for statistical analysis.Results The positive expression rate of CALL in ESCC tissues was 56 ％ (56/100),which was lower than that of tumor-adjacent normal tissues (95％,95/100),and the difference was statistically significant (x2=41.114,P＜0.01).There were statistically significant differences in CALL expression at protein level among patients with ESCC of different differentiation degree,different pathological T stage,lymph node metastasis and different TNM stage (x2=13.702,5.317,21.453,Fisher's exact test;all P＜ 0.05).The five year disease related survival rate of ESCC patients with down-regulated expression of CALL was 0(0/49),which was lower than those with normal CALL expression (25.5％,13/51),and the difference was statistically significant (x2 =43.338,P＜0.01).The median survival time of CALL expression down-regulated group was 17 months,and that of normal expressed group was 38 months.CALL expression was an independent risk factor of disease special survival rate (hazard ratio (HR) 0.353,95％ confidence interval (CI) 0.188 to 0.666,P=0.001).The results of wound-healing assay showed that the migration ability of CALL overexpressed CALL-k30 cells was lower than that of Vec-k30 cells in control group on 24 hours after wound.The results of Transwell invasion test showed the number of migrating cells penetrating CALL k30 cells attached to the inferior surface of the membrane was 44.000±13.748,which was less than that of the Vec k30 cells (154.333±25.007),and the difference was statistically significant (t=5.136,P=0.036).The results of F-actin staining demonstrated that actin filaments of CALL-k30 cells was 234.667 ± 65.118,which was lower than that of Vec-k30 cells (597.000± 119.929),and the difference was statistically significant (t=4.707,P=0.042).Conclusions CALL lowers the migration and invasion abilities of esophageal cancer cells by inhibiting F-actin microfilaments.Its abnormal expression may play an important role in the genesis,development and prognosis of ESCC.

Effects of dynamic pressure on the expression of PTHrP mRNA in metaphyseal cartilage stem cells of rats / 西安交通大学学报(医学版)

Objective To study the effect of dynamic pressure on the expression of parathyroid hormone-related protein (PTHrP)mRNA in metaphyseal cartilage stem cells of rats so as to further explore whether fiber actin (F-actin)is involved in the mechanical signal transduction process.Methods We isolated and cultured metaphyseal cartilage stem cells of rats by immunomagnetic beads.The third-generation rat metaphyseal cartilage stem cells were randomly divided into four groups:0%,3%,6%,and 12% deformed groups according to the size of dynamic pressure strength.We used a self-prepared dynamic tonic culture device to exert different intensity of pressure on each group of cells for 24 hours.Flow cytometry was used to detect the cell cycle distribution and apoptosis rate.The expression of PTHrP mRNA in each group was detected by Rea-l time quantitative PCR. Furthermore,the third-generation rat metaphyseal cartilage stem cells were randomly divided into four groups:control group,simple pressure group (6% deformation),pressure+cytoskeleton relaxin D group,and simple cytoskeleton relaxin D group according to whether or not to apply pressure and cytoskeleton relaxin D.F-actin fibers in each group of cells were stained with phalloidin and placed under a laser scanning confocal microscope.The expression of PTHrP mRNA in each group was detected by Real-time quantitative PCR.Results The results of flow cytometry showed no significant difference in G0/G1,G2/M and S phases between 0%,3%,6% and 12% deformed groups (P＞0.05).There was no significant difference in the apoptosis rate between 3% and 6% deformed groups compared with 0% deformed group (P＞0.05).The apoptosis rate was significantly higher in 1 2 % deformed group than in control group (P＜0.05).The results of laser confocal microscopy showed that the arrangement of F-actin fibers in the pressure group was neat and parallel compared with that in the control group, which was consistent with the direction of force.The intracellular F-actin fiber structure in pressure+cytoskeleton relaxin D group and simple cytoskeleton relaxin D group was destroyed and aggregated into clusters.Real-time quantitative PCR results showed that PTHrP mRNA expression did not significantly differ between 3% and 0% deformed groups (P＞0.05).The expression of PTHrP mRNA in 6% and 12% deformed groups was significantly higher than that in 0% group (P＜0.05).The expression of PTHrP mRNA in the cells of simple pressure group was significantly higher than that in the control group (P＜0.05).There was no significant difference in the expression of PTHrP mRNA between simple cytoskeleton relaxin D group and control group (P＞0.05).The mRNA expression of PTHrP was higher in pressure+cytoskeleton relaxin D group than that in control group,but lower than in simple pressure group (P＜0.05).Conclusion The dynamic pressure of proper intensity can increase the mRNA expression of PTHrP in chondrocytes of metaphyseal hypertrophy in rats,and F-actin is involved in the mechanical signal transduction process.

Effects of wild-type PTEN overexpression and its mutation on F-actin in activated hepatic stellate cells / 中华肝脏病杂志

Objective@#To investigate the effect of overexpression of wild-type phosphatase and tensin homolog (PTEN) deleted on chromosome 10 and its mutant G129E (exhibiting the activity of protein phosphatase and losing the activity of lipid phosphatase) on F-actin in activated hepatic stellate cells (HSCs) cultured in vitro.@*Methods@#The activated hepatic stellate cell-T6 (HSC-T6) cells were cultured in vitro, and activated HSCs were transfected with adenovirus that carried wild-type PTEN gene and G129E gene using transient transfection. The HSCs were divided into the following groups: control group, which was transfected with DMEM medium instead of virus solution; Ad-GFP group, which was transfected with the empty adenovirus vector with the expression of green fluorescent protein (GFP); Ad-PTEN group, which was transfected with the recombinant adenovirus with wild-type PTEN gene and GFP expression; Ad-G129E group, which was transfected with the recombinant adenovirus with G129E gene and GFP expression. Western blot and quantitative real-time PCR were used to measure the protein and mRNA expression of PTEN in activated HSCs; under a laser scanning confocal microscope (LSCM), phalloidine labeled with the fluorescein tetramethylrhodamine isothiocyanate (TRITC) was used to observe the morphology of HSCs, distribution and fluorescence intensity of F-actin, and changes in pseudopodia and stress fibers, and a calcium fluorescence probe (Rhod-2/AM) was used to measure the changes in Ca2+ concentration in HSCs. A one-way analysis of variance was used for comparison between multiple groups, and the least significant difference test was used for comparison between two groups.@*Results@#Wild-type PTEN and G129E genes were highly expressed in activated HSCs. In the control group and the Ad-GFP group, HSCs had a starlike or polygonal shape, F-actin was reconfigured and formed a large number of stress fibers which stretched across the whole cell, and layered pseudopodia were seen around the cell. In the Ad-PTEN group and the Ad-G129E group, the HSCs had a fusiform shape, F-actin was mainly seen around the cell, a small number of stress fibers were seen inside the cell, and layered pseudopodia around the cell disappeared. The Ad-PTEN group and the Ad-G129E group had significant reductions in the fluorescence intensity of F-actin compared with the control group and the Ad-GFP group (357.67±13.39/377.25±14.55 vs 961.87±27.33/954.68±20.71, F = 1783.486, P < 0.05), while there were no significant differences between the Ad-PTEN group and the Ad-G129E group, as well as between the control group and the Ad-GFP group (P > 0.05). The Ad-PTEN group and the Ad-G129E group had significant reductions in the relative concentration of Ca2+ compared with the control group and the Ad-GFP group (251.60±90.88/352.18±146.01 vs 1953.95±132.99/1937.57±115.17, F = 834.988, P < 0.05), while there were no significant differences between the Ad-PTEN group and the Ad-G129E group, as well as between the control group and the Ad-GFP group (P > 0.05).@*Conclusion@#The overexpressed wild-type PTEN and its mutant G129E can significantly inhibit the formation and reconfiguration of cytoskeletal protein F-actin and reduce the concentration of Ca2+ in activated HSCs in vitro. In addition, there are no significant differences in the above effects between wild-type PTEN and G129E.

Study on Rac1/MAPK/ERK pathway mediated mechanism and role in rats with ventilator induced lung injury / 中华危重病急救医学

Objective To investigate the role of Ras-related C3 botulinum toxin substrate 1/mitogen-activated protein kinase/extracellular signal-regulated kinase (Rac 1/MAPK/ERK) signal pathway in rats with ventilator induced lung injury (VILI) and its mechanism.Methods Thirty Sprague-Dawley (SD) rats were randomly divided into spontaneous respiration group,normal tidal volume (VT) group and high VT group with 10 rats in each group.The rats in spontaneous respiration group were kept their spontaneous breathing.The rats in normal VT group and high VT group were performed tracheal intubation after tracheostomy,and underwent mechanical ventilation on bilateral lungs with 6 mL/kg and 40 mL/kg VT respectively with maintenance anesthesia.After 4-hour ventilation,heart blood,bronchoalveolar lavage fluid (BALF) and lung tissues were harvested.The levels of interleukins (IL-1β,IL-6),tumor necrosis factor-α (TNF-α),myeloperoxidase (MPO) and macrophage inflammatory protein-2 (MIP-2) in serum and BALF were determined by enzyme linked immunosorbent assay (ELISA).Lung wet/dry radio (W/D) was determined.The lung tissues were stained with hematoxylin and eosin (HE),and pathological changes were observed,and pathological scores were evaluated.The ultra structure changes in type Ⅱ alveolar epithelial cells (AEC Ⅱ)were observed with transmission electron microscope.The positive expressions of phosphorylation of extracellular signal-regulated kinase (p-ERK) were determined by immunohistochemistry,and those of Racl and F-actin were determined by immunofluorescence.The mRNA expressions of ERK and Rac1 were determined by real-time fluorescent quantitation reverse transcription-polymerase chain reaction (RT-qPCR),and protein expressions of Rac-1,p-ERK and F-actin were determined by Western Blot.Results ① Compared with spontaneous breathing group,lung W/D in both mechanical ventilation groups was significantly increased,with more significant increase in the high VT group (6.64 ± 0.88 vs.1.79 ± 0.36,P ＜ 0.01).② There was no obvious pathological changes in the lung tissue and AEC Ⅱ of the spontaneously breathing group.In the normal VT group,there was slight edema and infiltration of inflammatory cells;AEC Ⅱ had less lamellar bodies and uniform distribution of the villi of the alveolar epithelium.In the high VT group,the edema of the lung tissue,the widening of the pulmonary septum,the alveolus congestion,the infiltration of inflammatory cells,and alveolar structure disorder were found;and AEC Ⅱ was irregular,the number of lamellar bodies in the plastids was decreased and was unevenly distributed.The pulmonary histopathological score in the high VT group was significantly higher than that in the spontaneous breathing group and the normal VT group (12.00 ± 2.00 vs.6.00 ± 1.51,8.50 ± 0.53,both P ＜ 0.01).③ Compared with spontaneous breathing group,IL-1β,IL-6,TNF-α,MPO,and MIP-2in serum and BALF in both mechanical ventilation groups were significantly increased,with more siguificant increase in the high VT group [serum IL-1 β (ng/L):104.2 ± 15.1 vs.20.3 ± 8.3,IL-6 (ng/L):46.6 ± 11.5 vs.22.7 ± 7.5,TNF-α (ng/L):39.4±6.5 vs.5.4± 1.9,MPO (ng/L):0.66±0.24 vs.0.06±0.03,MIP-2 (ng/L):109.2±25.8 vs.22.8±8.4;BALF IL-1 β (ng/L):121.5 ± 25.6 vs.24.0 ± 7.5,IL-6 (ng/L):136.7 ± 32.7 vs.31.4 ± 10.5,TNF-α (ng/L):98.0 ± 14.8vs.10.1 ±2.6,MPO (ng/L):0.80±0.31 vs.0.08±0.04,MIP-2 (ng/L):144.4±28.9 vs.41.2±20.7;all P ＜ 0.01].④ There were only a few p-ERK,Rac1 and F-actin positive expressions in the spontaneous breathing group.The positive expressions in normal VT group were increased.In high VT group,the positive expression of p-ERK was significantly increased;Rac1 and F-actin were mainly distributed in the cell membrane and cytoplasm respectively,the positive expressions were further enhanced.⑤ The gene expressions of ERK and Rac1,and protein expressions of p-ERK,Rac1 and F-actin in the high VT group were significantly higher than those in the spontaneous breathing group and normal VT group [ERK mRNA (2-△△Ct):8.23±2.83 vs.1,3.02± 1.38,p-ERK protein (gray value):1.15±0.36 vs.0.61 ±0.23,0.88±0.22;Rac1 mRNA (2-△△Ct):4.45 ±2.26 vs.1,1.22±0.39,Rac1 protein (gray value):0.91 ±0.16 vs.0.48±0.11,0.55 ± 0.10;F-actin protein (gray value):0.70± 0.09 vs.0.49 ± 0.08,0.55 ± 0.04;all P ＜ 0.01].Conclusion F-actin expression in lung tissue was up-regulated in rats with VILI,which resulted in reconstruction of AEC Ⅱ cyto-skeleton,and variation of cell membrane permeability through Rac 1 /MAPK/ERK sigualing pathway during VILI.

PKB/Akt regulates the aggregation of actin by Girdin in mouse fertilized eggs / 生物工程学报

The purpose of this study is to reveal the role of Girdin in regulating the aggregation of actin filaments by studying the relationship between PKB/Akt and Girdin. First we used Scansite software (http://scansite.mit.edu) to predict relevant target sites of PKB/Akt on mouse Girdin. To gain insight into the role of phosphorylation of Girdin by PKB/Akt, we assessed the location of phosphorylated Girdin in fertilized eggs by staining with anti-P-Girdin 1 417 Ab. We detected a distinct increase in the fluorescence signal of F-actin and P-Girdin 1 417 after microinjection of Akt WT and myr-Akt. The addition of myr-Akt induced phosphorylation of Girdin in mouse fertilized eggs. In addition, siRNA-mediated Akt-knockdown blocked phosphorylation of Girdin. The distribution of actin filaments was obviously scattered. These results strongly suggest that PKB/Akt could directly phosphorylate Girdin on Ser1 417 and promote its function in mouse fertilized eggs.

Yap1 plays a protective role in suppressing free fatty acid-induced apoptosis and promoting beta-cell survival

Mammalian pancreatic β-cells play a pivotal role in development and glucose homeostasis through the production and secretion of insulin. Functional failure or decrease in β-cell number leads to type 2 diabetes (T2D). Despite the physiological importance of β-cells, the viability of β-cells is often challenged mainly due to its poor ability to adapt to their changing microenvironment. One of the factors that negatively affect β-cell viability is high concentration of free fatty acids (FFAs) such as palmitate. In this work, we demonstrated that Yes-associated protein (Yap1) is activated when β-cells are treated with palmitate. Our loss- and gain-of-function analyses using rodent insulinoma cell lines revealed that Yap1 suppresses palmitate-induced apoptosis in β-cells without regulating their proliferation. We also found that upon palmitate treatment, re-arrangement of F-actin mediates Yap1 activation. Palmitate treatment increases expression of one of the Yap1 target genes, connective tissue growth factor (CTGF). Our gain-of-function analysis with CTGF suggests CTGF may be the downstream factor of Yap1 in the protective mechanism against FFA-induced apoptosis.

Plastins regulate ectoplasmic specialization via its actin bundling activity on microfilaments in the rat testis / 亚洲男科学杂志(英文版)

Plastins are a family of actin binding proteins (ABPs) known to cross-link actin microfilaments in mammalian cells, creating actin microfilament bundles necessary to confer cell polarity and cell shape. Plastins also support cell movement in response to changes in environment, involved in cell/tissue growth and development. They also confer plasticity to cells and tissues in response to infection or other pathological conditions (e.g., inflammation). In the testis, the cell-cell anchoring junction unique to the testis that is found at the Sertoli cell-cell interface at the blood-testis barrier (BTB) and at the Sertoli-spermatid (e.g., 8-19 spermatids in the rat testis) is the basal and the apical ectoplasmic specialization (ES), respectively. The ES is an F-actin-rich anchoring junction constituted most notably by actin microfilament bundles. A recent report using RNAi that specifically knocks down plastin 3 has yielded some insightful information regarding the mechanism by which plastin 3 regulates the status of actin microfilament bundles at the ES via its intrinsic actin filament bundling activity. Herein, we provide a brief review on the role of plastins in the testis in light of this report, which together with recent findings in the field, we propose a likely model by which plastins regulate ES function during the epithelial cycle of spermatogenesis via their intrinsic activity on actin microfilament organization in the rat testis.

Signaling mechanisms of glucose-induced F-actin remodeling in pancreatic islet beta cells

The maintenance of whole-body glucose homeostasis is critical for survival, and is controlled by the coordination of multiple organs and endocrine systems. Pancreatic islet beta cells secrete insulin in response to nutrient stimuli, and insulin then travels through the circulation promoting glucose uptake into insulin-responsive tissues such as liver, skeletal muscle and adipose. Many of the genes identified in human genome-wide association studies of diabetic individuals are directly associated with beta cell survival and function, giving credence to the idea that beta-cell dysfunction is central to the development of type 2 diabetes. As such, investigations into the mechanisms by which beta cells sense glucose and secrete insulin in a regulated manner are a major focus of current diabetes research. In particular, recent discoveries of the detailed role and requirements for reorganization/remodeling of filamentous actin (F-actin) in the regulation of insulin release from the beta cell have appeared at the forefront of islet function research, having lapsed in prior years due to technical limitations. Recent advances in live-cell imaging and specialized reagents have revealed localized F-actin remodeling to be a requisite for the normal biphasic pattern of nutrient-stimulated insulin secretion. This review will provide an historical look at the emergent focus on the role of the actin cytoskeleton and its regulation of insulin secretion, leading up to the cutting-edge research in progress in the field today.

The role of p38MAPK in asymmetric dimethylarginineinduced cytoskeleton and cellular permeability changes in cultured endothelial cells.

Background: Asymmetric dimethylarginine (ADMA) induces endothelial cell barrier dysfunction via cytoskeleton activation and contraction. It is supposed that activated p38 mitogen-activated protein kinase (MAPK) would trigger the formation of stress fibers and increase cellular permeability. Objective: Explore p38 MAPK as a potentially important enzyme in ADMA-mediated endothelial cell contractile response and permeability change. Methods: Human umbilical endothelial cells (HUVECs) were cultured, where ADMA and/or SB203580 (the specific inhibitor of p38MAPK) were used to stimulate HUVECs. Immunofluorescent staining was carried out to examine the expression and distribution of F-actin, flow cytometry was used to quantify F-actin, and Transwell was applied to test cellular permeability with FITC-labelled human serum albumin (HSA). Scanning electronic microscopy (SEM) was utilized to observe the changes of intercellar contact. Results: ADMA induced significant p38MAPK activation in a dose-dependent manner, which correlated with increased stress fibers. SB-203580 attenuated the formation of actin stress fiber and the increase of cellular permeability induced ADMA in the HUVECs (p<0.01, LSCM; p<0.01, cytometry; p<0.05, Transwell). Widened intercellular space induced by ADMA was detected and could be inhibited by SB-203580 (SEM). SB-203580 alone had no effect on cytoskeleton and cellular permeability. Conclusion: p38MAPK activation participated in cytoskeleton and cellular permeability changes induced by ADMA in HUVECs.

Characterization of the Rho GTPase-Activating Protein RhoGAP68F

Rho small GTPases control multiple aspects of neuronal morphogenesis by regulating the assembly and organization of the actin cytoskeleton. Although they are negatively regulated by GTPase activating proteins (GAPs), the roles of RhoGAPs in the nervous system have not been fully investigated. Here we describe a characterization of Drosophila RhoGAP68F that is mainly expressed in the embryonic central nervous system. RNA in situ hybridization analysis showed that expression of RhoGAP68F is highly restricted to the embryonic brain and ventral nerve cord. Database search revealed that RhoGAP68F contains an N-terminal Sec14 domain and a C-terminal RhoGAP domain. Rho-GTP pull-down assay demonstrated that the RhoGAP domain of RhoGAP68F inactivates RhoA but not Rac1 or Cdc42 in HEK293 cells. In addition, expression of RhoGAP68F in NIH3T3 cells suppressed LPA-induced stress fiber formation, which is mediated by RhoA. Finally, neuronal overexpression of RhoGAP68F causes synaptic overgrowth at the larval neuromuscular junction (NMJ). Taken together, these results suggest that RhoGAP68F may play a role in synaptic growth regulation by inactivating RhoA.

Effects of endothelin-1 on the cytoskeleton protein F-actin of human trabecular meshwork cells in vitro / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To observe the effect of endothelin-1(ET-1) on the cytoskeleton protein F-actin of cultured human trabecular meshwork (HTM) cells. METHODS: Cultured HTM cells were randomly divided into four groups: control group(0mol/L), low-dose ET-1(10-9mol/L) treatment group, middle-dose ET-1(10-8 mol/L) treatment group, and high-dose ET-1(10-7 mol/L) treatment group. After treated with ET-1, the expression of cytoskeleton protein F-actin in trabecular meshwork was analyzed with Western-blot and the distribution of F-actin was detected with FITC-Phalloidin probe. RESULTS: ET-1 dose-dependently and significantly increased F-actin in trabecular meshwork cells. The F-actin stress fiber and periphery actin fiber highly increased and manifested mild reorganization after treated with ET-1; and there were much more cell-to-cell and cell-to-extracellular matrix attachments formation in ET-1 treated HTM cells than that in the untreated HTM cells. CONCLUSION: ET-1 promoted the expression of cytoskeleton protein F-actin and induced the trabecular meshwork actin cytoskeleton reorganization.