The adult musculature of two pseudostomid species reveals unique patterns for flatworms (Platyhelminthes, Prolecithophora).

We analyzed the adult musculature of two prolecithophoran species, Cylindrostoma monotrochum (von Graff, 1882) and Monoophorum striatum (von Graff, 1878) using a phalloidin-rhodamine technique. As in all rhabdithophoran flatworms, the body-wall musculature consisted of three muscle layers: on the outer side was a layer of circular muscle fibers and on the inner side was a layer of longitudinal muscle fibers; between them were two different types of diagonally orientated fibers, which is unusual for flatworms. The musculature of the pharynx consisted of a basket-shaped grid of thin longitudinal and circular fibers. Thick anchoring muscle fibers forming a petal-like shape connected the proximal parts of the pharynx with the body-wall musculature. Male genital organs consisted of paired seminal vesicles, a granular vesicle, and an invaginated penis. Peculiar ring-shaped muscles were only found in M. striatum, predominantly in the anterior body part. In the same species, seminal vesicles and penis only had circular musculature, while in C. monotrochum also longitudinal musculature was found in these organs. Female genital organs were only present in M. striatum, where we characterized a vagina interna, and a bursa seminalis. Transverse, crossover, and dorsoventral muscle fibers were lacking in the middle of the body and greatly varied in number and position in both species.

[Effects of denatured collagen type â on differentiation of human fibroblasts into myofibroblasts].

Objective: To investigate the effects of denatured collagen type Ⅰ on differentiation of human fibroblasts into myofibroblasts. Methods: A small amount of normal skin donated by burn patients undergoing scar surgery was collected. Human fibroblasts were obtained by method of explant culture and then sub-cultured. The fourth passage of cells were used in the following experiments. (1) Fibroblasts were divided into normal collagen group and denatured collagen group according to the random number table, with 10 wells in each group. Fibroblasts in normal collagen group were cultured on normal collagen type Ⅰ coated coverslips. Fibroblasts in denatured collagen group were cultured on denatured type Ⅰ collagen coated coverslips. Expression of proliferating cell nuclear antigen (PCNA) was detected by immunohistochemical method, and the percentage of PCNA positive cells was calculated. (2) Another batch of fibroblasts were grouped and treated as in (1), with 12 wells in each group. Proliferation activity of cells was determined with methyl-thiazolyl-tetrazolium colorimetry method. (3) Another batch of fibroblasts were grouped and treated as in (1), and the microfilament morphology of cells was observed by rhodamine-phalloidin staining. (4) Another batch of fibroblasts were grouped and treated as in (1). Expression of α smooth muscle actin (α-SMA) of cells was detected by immunohistochemical method, and expression of OB-cadherin of cells was detected by immunofluorescence method. (5) Another batch of fibroblasts were divided into normal collagen, denatured collagen, and common coverslips groups according to the random number table, with 6 wells in each group. Fibroblasts in normal collagen and denatured collagen groups were treated as in (1), while fibroblasts in common coverslips group were cultured on coverslips without collagen coating. Expressions of α-SMA and OB-cadherin of cells were determined with Western blotting. (6) Another batch of fibroblasts were grouped and treated as in (5), and then the mRNA expressions of collagen type Ⅰ, collagen type Ⅲ, and α-SMA of cells were determined with real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were processed with t test, one way analysis of variance, and least-significant difference test. Results: (1) The percentage of PCNA positive cells in denatured collagen group was (83±9)%, significantly higher than (29±9)% in normal collagen group (t=13.53, P<0.01). (2) The proliferation activity of fibroblasts in denatured collagen group was 0.32±0.06, significantly higher than 0.25±0.05 in normal collagen group (t=3.06, P<0.01). (3) The microfilament of fibroblasts in normal collagen group was arranged vertically and in parallel way, paralleling the long axis of cells. The microfilament of fibroblasts in denatured collagen group was denser and thicker. (4) Most fibroblasts in normal collagen group showed long shuttle-like shape typically. Morphology of fibroblasts in denatured collagen group changed, and cells were obviously spreading. Expressions of α-SMA and OB-cadherin of fibroblasts in denatured collagen group were stronger than those in normal collagen group. (5) Expressions of α-SMA of fibroblasts in denatured collagen, normal collagen, and common coverslips groups were respectively 1.69±0.41, 0.89±0.27, and 1.46±0.42. Expression of α-SMA of fibroblasts in denatured collagen group was significantly higher than that in normal collagen group (P<0.01). Expressions of OB-cadherin of fibroblasts in denatured collagen, normal collagen, and common coverslips groups were respectively 5.17±0.28, 2.21±0.10, and 4.01±0.56. Expression of OB-cadherin of fibroblasts in denatured group was significantly higher than that in normal collagen group (P<0.01). (6) There was no significant difference in mRNA expression of collagen type Ⅰ of fibroblasts in denatured collagen, normal collagen, and common coverslips groups (F=2.71, P>0.05). The mRNA expressions of collagen type Ⅲ and α-SMA of fibroblasts in normal collagen group were significantly lower than those in denatured collagen group (P<0.01). Conclusions: Denatured collagen type Ⅰ may influence the activity of fibroblasts, thus inducing fibroblasts differentiating into myofibroblasts.

Regulation of actin filament turnover by cofilin-1 and cytoplasmic tropomyosin isoforms.

Tropomyosin and cofilin are actin-binding proteins which control dynamics of actin assembly and disassembly. Tropomyosin isoforms can either inhibit or enhance cofilin activity, but the mechanism of this diverse regulation is not well understood. In this work mechanisms of actin dynamics regulation by four cytoskeletal tropomyosin isoforms and cofilin-1 were studied with the use of biochemical and fluorescent microscopy assays. The recombinant tropomyosin isoforms were products of two genes: TPM1 (Tpm1.6 and Tpm1.8) and TPM3 (Tpm3.2 and Tpm3.4). Tpm1.6/1.8 bound to F-actin with higher apparent binding constants and lower cooperativities than Tpm3.2/3.4. In consequence, subsaturating concentrations of cofilin-1 removed 50% of Tpm3.2/3.4 from F-actin. By contrast, 2 and 5.5 molar excess of cofilin-1 over actin was required to dissociate 50% of Tpm1.6/1.8. All tropomyosins inhibited the rate of spontaneous polymerization of actin, which was reversed by cofilin-1. Products of TPM1 favored longer filaments and protected them from cofilin-induced depolymerization. This was in contrast to the isoforms derived from TPM3, which facilitated depolymerization. Tpm3.4 was the only isoform, which increased frequency of the filament severing by cofilin-1. Tpm1.6/1.8 inhibited, but Tpm3.2/3.4 enhanced cofilin-induced conformational changes leading to accelerated release of rhodamine-phalloidin from the filament. We concluded that the effects were executed through different actin affinities of tropomyosin isoforms and cooperativities of tropomyosin and cofilin-1 binding. The results obtained in vitro were in good agreement with localization of tropomyosin isoforms in stable or highly dynamic filaments demonstrated before in various cells.

Study on adhesion, proliferation and differentiation of osteoblasts promoted by new absorbable bioactive glass injection in vitro.

OBJECTIVE: The objective of this study was to evaluate adhesion, proliferation and differentiation of osteoblasts grown on absorbable bioactive glass-calcium phosphate cement injection (BG-CPC) materials in vitro. MATERIALS AND METHODS: BG-CPC composite biomaterial samples were prepared in vitro, for culture with MC3T3-E1 rat osteoblasts. Cells were divided into CPC, BG and BG-CPC treated groups. After cultivation for 3d, cells were stained with rhodamine-phalloidin and 4',6-diamidino-2-phenylindole (DAPI) and observed by fluorescence microscopy for osteoblast morphology on the surface of biomaterials. At 24h, 48h and 72h, MTT assay was used to test adhesion and proliferation, and bicinchoninic acid assay (BCA) method was carried out to test ALP activity; ELISA was used to test bone morphogenetic protein (BMP) and TGF-ß expression levels at day 3. RESULTS: Compared with the other two groups, cells in the BG-CPC group had more attachments; the DAPI labelled nuclei were clearer and nuclear shape was more complete and full. Adhesion and proliferation, as well as alkaline phosphatase (ALP) activity of cells for all time points in the BG-CPC group were higher than those in the other two groups and differences were of statistically significant (p<0.05); BMP and TGF-ß expression levels were higher than those in the other two groups and the differences were statistically significant (p<0.05). CONCLUSIONS: In vitro use of new absorbable bioactive glass is able to promote adhesion, proliferation and differentiation of osteoblasts, which may be related to increased BMP and TGF-ß expression.

[Effects of transforming growth factor ß1 receptor inhibitor SD-208 on human hypertrophic scar].

OBJECTIVE: To investigate the effects of transforming growth factor ß1 (TGF-ß1) receptor inhibitor SD-208 on human hypertrophic scar and its mechanisms. METHODS: Scar fibroblasts were isolated from deprecated human hypertrophic scar tissue and then sub-cultured. Cells of the fifth passage were used in the following experiments. (1) Cells were divided into blank control group (BC) and 0.5, 1.0, 3.0, and 5.0 µmol/L SD-208 groups according to the random number table (the same grouping method below), with 6 wells in each group. Cells in group BC were added with 1 µL phosphate buffer solution, while cells in the latter four groups were added with 0.5, 1.0, 3.0, and 5.0 µmol/L SD-208, respectively. After being cultured for 12 hours, the proliferation activity of cells was detected by cell counting kit 8 and microplate reader (denoted as absorbance value). Suitable amount of substance concentration of SD-208 according to the results of proliferation activity of cells was chosen for the following experiments. (2) Another batch of cells were divided into group BC and 1, 3 µmol/L SD-208 groups and treated as in (1), with 8 wells in each group. The number of migration cells was detected by transwell method. (3) Another batch of cells were grouped and treated as in (2), and the microfilament morphology of cells was observed by rhodamine-phalloidin staining. (4) Another batch of cells were grouped and treated as in (2), and the protein expression of TGF-ß1 was assessed with Western blotting. (5) Forty-eight BALB/c nude mice were divided into normal saline group (NS) and 1 µmol/L SD-208 group, and one longitudinal incision with length of 1 cm was made on their back. Then human hypertrophic scar tissue was embedded into the incision. On post injury day 7, multipoint injection of NS in a volume of 0.05 mL was performed in wounds of rats in group NS, while rats in 1 µmol/L SD-208 group were given 0.05 mL 1 µmol/L SD-208, once a day. On the day 0 (the same day), 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 post first time of injection, the weight of 8 nude mice was weighed by electronic scale, and scar area was measured by vernier caliper and the ratio of rest scar area was calculated. (6) In week 1, 2, and 3 post first time of injection, the protein expression of TGF-ß1 of human hypertrophic scar tissue was assessed with Western blotting. Data were processed with one-way analysis of variance and two independent-sample t test. RESULTS: (1) The proliferation activity of cells in group BC, 0.5, 1.0, 3.0, and 5.0 µmol/L SD-208 groups was respectively 1.00±0.03, 0.90±0.08, 0.68±0.11, 0.54±0.04, and 0.42±0.09, and the proliferation activity of cells in 0.5, 1.0, 3.0, and 5.0 µmol/L SD-208 groups was significantly lower than that in group BC (with t values from 2.9 to 22.1, P<0.05 or P<0.01). (2) The number of migration cells in 1, 3 µmol/L SD-208 groups was significantly less than that in group BC (with t values respectively 6.5 and 6.4, P values below 0.01). (3) Compared with that in group BC, fluorescence intensity of microfilaments of cells in 1, 3 µmol/L SD-208 groups was attenuated, and the pseudopod extended less. (4) The protein expressions of TGF-ß1 of cells in group BC and 1, 3 µmol/L SD-208 groups were respectively 1.00±0.08, 0.80±0.08, and 0.61±0.05, and the protein expressions of TGF-ß1 of cells in 1, 3 µmol/L SD-208 groups were significantly lower than those in group BC (with t values respectively 4.0 and 9.2, P values below 0.01). (5) The weights of nude mice in group NS and 1 µmol/L SD-208 group were similar on each time day (with t values from 0.2 to 1.1, P values above 0.05). The ratios of rest scar area of nude mice in two groups were decreased along with the injection time, and the ratios of rest scar area of nude mice in 1 µmol/L SD-208 group were significantly less than those in group NS from the day 6 to 20 post first time of injection (with t values from 1.8 to 15.9, P<0.05 or P<0.01). In week 1, 2, and 3 post first time of injection, the protein expressions of TGF-ß1 of human hypertrophic scar tissue in nude mice in two groups showed a tendency of decrease, and the protein expressions of TGF-ß1 of human hypertrophic scar tissue in nude mice in 1 µmol/L SD-208 group were significantly lower than those in group NS (with t values from 6.2 to 19.1, P values below 0.01). CONCLUSIONS: SD-208 has significant inhibition effect on human hypertrophic scars, and the mechanism is correlated to the inhibition of protein expression of endogenous TGF-ß1.

Impact of C24:0 on actin-microtubule interaction in human neuronal SK-N-BE cells: evaluation by FRET confocal spectral imaging microscopy after dual staining with rhodamine-phalloidin and tubulin tracker green.

Disorganization of the cytoskeleton of neurons has major consequences on the transport of neurotransmitters via the microtubule network. The interaction of cytoskeleton proteins (actin and tubulin) was studied in neuronal SK-N-BE cells treated with tetracosanoic acid (C24:0), which is cytotoxic and increased in Alzheimer's disease patients. When SK-N-BE cells were treated with C24:0, mitochondrial dysfunctions and a non-apoptotic mode of cell death were observed. Fluorescence microscopy revealed shrunken cells with perinuclear condensation of actin and tubulin. Impact of C24:0 on actin-microtubule interaction in human neuronal SK-N-BE cells: evaluation by FRET confocal spectral imaging microscopy after dual staining with rhodamine-phalloidin and tubulin tracker green After staining with rhodamine-phalloidin and with an antibody raised against α-/ß-tubulin, modifications of F-actin and α-/ß-tubulin levels were detected by flow cytometry. Lower levels of α-tubulin were found by Western blotting. In C24:0-treated cells, spectral analysis and fluorescence recovery after photobleaching (FRAP) measured by confocal microscopy proved the existence of fluorescence resonance energy transfer (FRET) when actin and tubulin were stained with tubulin tracker and rhodamine-phalloidin demonstrating actin and tubulin co-localization/interaction. In control cells, no FRET was observed. Our data demonstrate quantitative changes in actin and tubulin, and modified interactions between actin and tubulin in SK-N-BE cells treated with C24:0. They also show that FRET confocal imaging microscopy is an interesting method for specifying the impact of cytotoxic compounds on cytoskeleton proteins.

Amatoxin and phallotoxin concentration in Amanita phalloides spores and tissues.

Most of the fatal cases of mushroom poisoning are caused by Amanita phalloides. The amount of toxin in mushroom varies according to climate and environmental conditions. The aim of this study is to measure α-, ß-, and γ-amanitin with phalloidin and phallacidin toxin concentrations. Six pieces of A. phalloides mushrooms were gathered from a wooded area of Düzce, Turkey, on November 23, 2011. The mushrooms were broken into pieces as spores, mycelium, pileus, gills, stipe, and volva. α-, ß-, and γ-Amanitin with phalloidin and phallacidin were analyzed using reversed-phase high-performance liquid chromatography. As a mobile phase, 50 mM ammonium acetate + acetonitrile (90 + 10, v/v) was used with a flow rate of 1 mL/min. C18 reverse phase column (150 × 4.6 mm; 5 µm particle) was used. The least amount of γ-amanitin toxins was found at the mycelium. The other toxins found to be in the least amount turned out to be the ones at the spores. The maximum amounts of amatoxins and phallotoxin were found at gills and pileus, respectively. In this study, the amount of toxin in the spores of A. phalloides was published for the first time, and this study is pioneering to deal with the amount of toxin in mushrooms grown in Turkey.

The actin cytoskeleton organization and disorganization properties of the photosynthetic dinoflagellate Symbiodinium kawagutii in culture.

The actin cytoskeleton organization in symbiotic marine dinoflagellates is largely undescribed; most likely, due to their intense pigment autofluorescence and cell walls that block fluorescent probe access. Using a freeze-fracture and fixation procedure, we observed the actin cytoskeleton of Symbiodinium kawagutii cultured in vitro with fluorescently labeled phalloidin and by indirect immunofluorescence with monoclonal antibodies specific for actin. The cytoskeleton appeared as an organized network with interconnected cortical and cytoplasmic thick filaments, along with some intertwined fine filaments. It showed a grid-type, reticular pattern organized in a lattice-like structure within the cell and throughout the cytoplasm. This organization was similar when the observations were done with either fluorescein isothiocyanate (FITC)-phalloidin or anti-actin, although the latter showed a more evenly distributed fluorescence characteristic of nonpolymerized actin. The network organization collapsed upon treatment with latrunculin, resulting in bright foci and diffuse fluorescence. A similar effect was obtained upon butanedione monoxime treatment, except that no bright foci were observed. We have been able to successfully visualize the actin cytoskeleton of S. kawagutii cells using fluorescence-based procedures. This is the first report on the visualization of the organization of the actin cytoskeleton under various conditions in these walled, highly autofluorescent cells.

Simvastatin induces apoptosis and disruption of the actin cytoskeleton in human dental pulp cells and periodontal ligament fibroblasts.

OBJECTIVE: Simvastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, and widely used as cholesterol-lowering agent, has been suggested for its beneficial effects on alveolar bone formation, regeneration of dental pulp tissue and periodontal ligament. High doses of simvastatin appear to induce apoptosis in several cell types, but little is known about its possible effect on tooth-associated cells. Therefore, the effects of simvastatin were studied on apoptosis and cell morphology of human dental pulp cells (HDPCs) and periodontal ligament fibroblasts (HPLFs). METHODS: HDPCs/HPLFs obtained from 4 patients were cultured with or without various concentrations of simvastatin (0.1, 1, and 10µM) for 24, 48, and 72h. The 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate cell viability. The levels of apoptosis of HDPCs and HPLFs were measured by flow cytometry after Annexin V/propidium iodide double staining. Phalloidin-FITC and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining was used to examine differences in the actin cytoskeleton and nuclear morphology, respectively. RESULTS: The viability of HDPCs and HPLFs was significantly reduced after simvastatin treatment in a dose- and time-dependent manner (p<0.05). The apoptosis of HDPCs and HPLFs was significantly increased in 10µM simvastatin-treated cells (p<0.05). The effect on apoptosis was comparable for HDPCs and HPLFs. Nuclear staining showed typical apoptotic nuclear condensation and fragmentation in simvastatin-treated HDPCs/HPLFs. A dose- and time-dependent simvastatin-induced disruption of the actin cytoskeleton was observed in both cell types. CONCLUSION: Our data demonstrated that simvastatin decreases the viability of HDPCs and HPLFs, probably by inducing apoptosis.

A cotton LIM domain-containing protein (GhWLIM5) is involved in bundling actin filaments.

LIM-domain proteins play important roles in cellular processes in eukaryotes. In this study, a LIM protein gene, GhWLIM5, was identified in cotton. Quantitative RT-PCR analysis showed that GhWLIM5 was expressed widely in different cotton tissues and had a peak in expression during fiber elongation. GFP fluorescence assay revealed that cotton cells expressing GhWLIM5:eGFP fusion gene displayed a network distribution of eGFP fluorescence, suggesting that GhWLIM5 protein is mainly localized to the cell cytoskeleton. When GhWLIM5:eGFP transformed cells were stained with rhodamine-phalloidin there was consistent overlap in eGFP and rhodamine-palloidin signals, demonstrating that GhWLIM5 protein is colocalized with the F-actin cytoskeleton. In addition, high-speed cosedimentation assay verified that GhWLIM5 directly bound actin filaments, while low cosedimentation assay and microscopic observation indicated that GhWLIM5 bundled F-actin in vitro. Increasing amounts of GhWLIM5 protein were able to protect F-actin from depolymerization in vitro in the presence of Lat B (an F-actin depolymerizer). Our results contribute to a better understanding of the biochemical role of GhWLIM5 in modulating the dynamic F-actin network in cotton.

The Qdot-labeled actin super-resolution motility assay measures low-duty cycle muscle myosin step size.

Myosin powers contraction in heart and skeletal muscle and is a leading target for mutations implicated in inheritable muscle diseases. During contraction, myosin transduces ATP free energy into the work of muscle shortening against resisting force. Muscle shortening involves relative sliding of myosin and actin filaments. Skeletal actin filaments were fluorescently labeled with a streptavidin conjugate quantum dot (Qdot) binding biotin-phalloidin on actin. Single Qdots were imaged in time with total internal reflection fluorescence microscopy and then spatially localized to 1-3 nm using a super-resolution algorithm as they translated with actin over a surface coated with skeletal heavy meromyosin (sHMM) or full-length ß-cardiac myosin (MYH7). The average Qdot-actin velocity matches measurements with rhodamine-phalloidin-labeled actin. The sHMM Qdot-actin velocity histogram contains low-velocity events corresponding to actin translation in quantized steps of ~5 nm. The MYH7 velocity histogram has quantized steps at 3 and 8 nm in addition to 5 nm and larger compliance compared to that of sHMM depending on the MYH7 surface concentration. Low-duty cycle skeletal and cardiac myosin present challenges for a single-molecule assay because actomyosin dissociates quickly and the freely moving element diffuses away. The in vitro motility assay has modestly more actomyosin interactions, and methylcellulose inhibited diffusion to sustain the complex while preserving a subset of encounters that do not overlap in time on a single actin filament. A single myosin step is isolated in time and space and then characterized using super-resolution. The approach provides a quick, quantitative, and inexpensive step size measurement for low-duty cycle muscle myosin.

A new F-actin structure in fungi: actin ring formation around the cell nucleus of Cryptococcus neoformans.

The F-actin cytoskeleton of Cryptococcus neoformans is known to comprise actin cables, cortical patches and cytokinetic ring. Here, we describe a new F-actin structure in fungi, a perinuclear F-actin collar ring around the cell nucleus, by fluorescent microscopic imaging of rhodamine phalloidin-stained F-actin. Perinuclear F-actin rings form in Cryptococcus neoformans treated with the microtubule inhibitor Nocodazole or with the drug solvent dimethyl sulfoxide (DMSO) or grown in yeast extract peptone dextrose (YEPD) medium, but they are absent in cells treated with Latrunculin A. Perinuclear F-actin rings may function as 'funicular cabin' for the cell nucleus, and actin cables as intracellular 'funicular' suspending nucleus in the central position in the cell and moving nucleus along the polarity axis along actin cables.

Depolymerization of actin facilitates memory formation in an insect.

In mammals, memory formation and stabilization requires polymerization of actin. Here, we show that, in the honeybee, inhibition of actin polymerization within the brain centres involved in memory formation, the mushroom bodies (MBs), enhances associative olfactory memory. Local application of inhibitors of actin polymerization (Cytochalasin D or Latrunculin A) to the MBs 1 h before induction of long-term memory increased memory retention 2 and 24 h after the onset of training. Post-training application of Cytochalasin D also enhanced retention, indicating that memory consolidation is facilitated by actin depolymerization. We conclude that certain aspects of memory mechanisms could have been established independently in mammals and insects.

[The involvement of actin cytoskeleton in glutoxim and molixan effect on intracellular Ca(2+)-concentration in macrophages].

Glutoxim and molixan belong to new generation of disulfide-containing drugs with immunomodulatory, hepatoprotective and hemopoetic effect on cells. Using Fura-2AM microfluorimetry, two structurally distinct actin filament disrupters, latrunculin B and cytochalasin D, and calyculin A, which causes actin filaments condensation under plasmalemma, we have shown the involvement of actin cytoskeleton in the intracellular Ca(2+)-concentration increase induced by glutoxim or molixan in rat peritoneal macrophages. Morphological data obtained with the use of rhodamine-phalloidine have demonstrated that glutoxim and molixan cause the actin cytoskeleton reorganization in rat peritoneal macrophages.

Translucent titanium coating facilitates observation of osteoblast migration behavior on a titanium surface.

PURPOSE: The appropriate surface composition and topography are crucial for osseointegration of titanium dental implants, and surface properties are known to enhance cell adhesion and promote expression of specific osteoblastic genes. In this study, a translucent titanium coating on glass coverslip (TiGlass) was introduced as a potential tool for direct observation of cell behavior on a titanium surface. MATERIALS AND METHODS: Scanning electron microscopy, energy-dispersive x-ray analysis, and atomic force microscopy were performed on TiGlass to provide information about its physical properties. Random migration, osteoblastic gene expression, and immunofluorescence cell staining on TiGlass were also examined and analyzed. RESULTS: The translucent titanium surface offered excellent optical characteristics that facilitated transmitted light observations under an optical microscope, transforming the opaque metal into an observable titanium matrix. Random migration analysis of the primary osteoblasts on TiGlass revealed that the titanium coating enhanced the migration speed of the osteoblasts and significantly shortened the time lag for the initial migration behavior. Further study of osteoblastic gene expression on this smooth titanium surface revealed no significant changes. Co-localization of actin filament and vinculin was found on TiGlass under epifluorescent microscopy. CONCLUSION: The application of a translucent titanium-coated coverslip in vitro altered the migration pattern of osteoblasts. Collectively, the results suggest that titanium promotes initial adhesion and accelerates osteoblast migration.

The difference of fibroblast behavior on titanium substrata with different surface characteristics.

Connective tissue, one of the main components of peri-implant soft tissue, is key to the formation of the peri-implant mucosal seal and helping to prevent epithelial ingrowth. Rough surfaces (Rs), machined surfaces (Ms) or microgrooved surface (MG) are used in the neck area of commercially available titanium implants. In this paper, we aimed to evaluate the influence of surface topography of titanium substratum on connective tissue fibroblasts to gain a better understanding of this effect. Fibroblasts were cultured on titanium plates with Rs, Ms and MG. Adhesion cell number at day 3 was compared and protein distribution of both F-actin and vinculin was determined to observe cellular structure and adhesion. Cell adhesion strength was compared on each surface. At day 3, the number of fibroblasts attached on each substratum was in the order of MG ≈ Ms > Rs. Fibroblasts strongly expressed vinculin in the peripheral area on Ms and MG, and showed strong F-actin architecture. Decreased expression of vinculin and weaker continuity of F-actin were observed on Rs. Fibroblasts on MG were aligned along the grooves, with a significantly higher cell density, whereas cells on Ms and Rs had no clear orientation. The cell adhesion strength was significantly lower on Rs, and no significant difference was seen between MG and Ms. Both MG and Ms showed greater adhesion cell numbers and adhesion strength of fibroblasts when compared with Rs at day 3. The cell density on MG was greater than those on other substrata.

Enamel matrix protein adsorption to root surfaces in the presence or absence of human blood.

BACKGROUND: The clinical use of an enamel matrix derivative (EMD) has been shown to promote formation of new cementum, periodontal ligament (PDL), and bone and to significantly enhance the clinical outcomes after regenerative periodontal surgery. It is currently unknown to what extent the bleeding during periodontal surgery may compete with EMD adsorption to root surfaces. The aim of this study is to evaluate the effect of blood interactions on EMD adsorption to root surfaces mimicking various clinical settings and to test their ability to influence human PDL cell attachment and proliferation. METHODS: Teeth extracted for orthodontic reasons were subjected to ex vivo scaling and root planing and treated with 24% EDTA, EMD, and/or human blood in six clinically related settings to determine the ability of EMD to adsorb to root surfaces. Surfaces were analyzed for protein adsorption via scanning electron microscopy and immunohistochemical staining with an anti-EMD antibody. Primary human PDL cells were seeded on root surfaces and quantified for cell attachment and cell proliferation. RESULTS: Plasma proteins from blood samples altered the ability of EMD to adsorb to root surfaces on human teeth. Samples coated with EMD lacking blood demonstrated a consistent even layer of EMD adsorption to the root surface. In vitro experiments with PDL cells demonstrated improved cell attachment and proliferation in all samples coated with EMD (irrespective of EDTA) when compared to samples containing human blood. CONCLUSION: Based on these findings, it is advised to minimize blood interactions during periodontal surgeries to allow better adsorption of EMD to root surfaces.

Stretching actin filaments within cells enhances their affinity for the myosin II motor domain.

To test the hypothesis that the myosin II motor domain (S1) preferentially binds to specific subsets of actin filaments in vivo, we expressed GFP-fused S1 with mutations that enhanced its affinity for actin in Dictyostelium cells. Consistent with the hypothesis, the GFP-S1 mutants were localized along specific portions of the cell cortex. Comparison with rhodamine-phalloidin staining in fixed cells demonstrated that the GFP-S1 probes preferentially bound to actin filaments in the rear cortex and cleavage furrows, where actin filaments are stretched by interaction with endogenous myosin II filaments. The GFP-S1 probes were similarly enriched in the cortex stretched passively by traction forces in the absence of myosin II or by external forces using a microcapillary. The preferential binding of GFP-S1 mutants to stretched actin filaments did not depend on cortexillin I or PTEN, two proteins previously implicated in the recruitment of myosin II filaments to stretched cortex. These results suggested that it is the stretching of the actin filaments itself that increases their affinity for the myosin II motor domain. In contrast, the GFP-fused myosin I motor domain did not localize to stretched actin filaments, which suggests different preferences of the motor domains for different structures of actin filaments play a role in distinct intracellular localizations of myosin I and II. We propose a scheme in which the stretching of actin filaments, the preferential binding of myosin II filaments to stretched actin filaments, and myosin II-dependent contraction form a positive feedback loop that contributes to the stabilization of cell polarity and to the responsiveness of the cells to external mechanical stimuli.

[Fibrillar actin in the nuclear apparatus of the ciliate Paramecium caudatum].

Due to their nuclear dualism, ciliates provide a good model for studying the role of actin in spatial organization and transcription activity of the nucleus. The actin in the nuclear apparatus of the ciliate Paramecium caudatum was studied using fluorescently labeled phalloiodin and indirect immunocytochemistry. Fibrillar actin was demonstrated in both of the nuclei. Actin was revealed in the chromatin areas, and was often associated with the periphery of the amplified nucleoli in the macronucleus. Redistribution of actin was observed depending on different physiological state of the cells. Stable infection of the macronulear with the intranuclear endobionts Holospora obtuse led to the loss of nuclear actin accompanied by significant nuclear fragility and redistribution of the phosphorylated form of the actin-binding protein cofilin. Spherical bodies resembling karyosphere were found in the macronuclear anlagen.

Chick amniogenesis is mediated by an actin cable.

This study examined the process of chick amniogenesis to determine whether the actin cable mechanism operating during amnion wound repair is a recapitulation of developmental events. Staining of the developing amnion with fluorescein isothiocyanate-labeled phalloidin indicated the presence of an actin cable in the amniotic head fold, which persisted through to the closure of the amnion. Transmission electron microscopy of the developing amnion revealed linearly arranged actin microfilaments in the elongated cells at the leading edge of the amnion, adjacent to either side of a nodule of numerous cells at the point of midline fusion. A mesh of cytoplasmic actin filaments was seen dispersed throughout the accumulated cells of the nodule. Lamellapodia were absent suggesting that cell crawling is not involved in amniogenesis. Addition of an enzyme inhibitor of Rho, cell-permeable C3 transferase, to the surface of the developing amnion prior to closure appeared to inhibit amniogenesis at the early embryonic stages.