Polymerized Laminin-332 Matrix Supports Rapid and Tight Adhesion of Keratinocytes, Suppressing Cell Migration

Laminin-332 (α3ß3γ2) (Lm332) supports the stable anchoring of basal keratinocytes to the epidermal basement membrane, while it functions as a motility factor for wound healing and cancer invasion. To understand these contrasting activities of Lm332, we investigated Lm332 matrices deposited by normal human keratinocytes and other Lm332-expressing cell lines. All types of the cells efficiently deposited Lm332 on the culture plates in specific patterns. On the contrary, laminins containing laminin ß1 and/or γ1 chains, such as Lm511 and Lm311, were not deposited on the culture plates even if secreted into culture medium. The Lm332 deposition was not inhibited by function-blocking antibodies to the α3 and α6 integrins but was inhibited by sodium selenate, suggesting that sulfated glycosaminoglycans on cell surface, e.g. heparan sulfate proteoglycans, might be involved in the process. HEK293 cells overexpressing exogenous Lm332 (Lm332-HEK) almost exclusively deposited Lm332 on the plates. The deposited Lm332 matrix showed a mesh-like network structure as analyzed by electron microscopy, suggesting that Lm332 was highly polymerized. When biological activity was analyzed, the Lm332 matrix rather suppressed the migration of keratinocytes as compared with purified Lm332, which highly promoted the cell migration. The Lm332 matrix supported adhesion of keratinocytes much more strongly and stably than purified Lm332. Integrin α3ß1 bound to the Lm332 matrix at a three times higher level than purified Lm332. Normal keratinocytes prominently showed integrin α6ß4-containing, hemidesmosome-like structures on the Lm332 matrix but not on the purified one. These results indicate that the polymerized Lm332 matrix supports stable cell adhesion by interacting with both integrin α6ß4 and α3ß1, whereas unassembled soluble Lm332 supports cell migration

Controlling exchange bias in Fe3O4/FeO composite particles prepared by pulsed laser irradiation

Spherical iron oxide nanocomposite particles composed of magnetite and wustite have been successfully synthesized using a novel method of pulsed laser irradiation in ethyl acetate. Both the size and the composition of nanocomposite particles are controlled by laser irradiation condition. Through tuning the laser fluence, the Fe3O4/FeO phase ratio can be precisely controlled, and the magnetic properties of final products can also be regulated. This work presents a successful example of the fabrication of ferro (ferri) (FM)/antiferromagnetic (AFM) systems with high chemical stability. The results show this novel simple method as widely extendable to various FM/AFM nanocomposite systems

A case of accidental ingestion of a partial denture base removed by upper gastrointestinal endoscopy

Summary.The majority of accidental ingestions and aspirations of dental foreign bodies occur during dental treatment.However,most of them are naturally excreted without any problem although the time to excretion varies.Among dental foreign bodies,removable partial dentures involve the risk of causing perforation of the digestive tract due to the morphology of the clasp;therefore,improper treatment or measures may cause serious complications.Now,there are increasing opportunities to proactively use an endoscope to remove foreignsubstances from the gastrointestinal tract.We report a case of accidental ingestion of a removable partial denture,extracted by upper gastrointestinal endoscopy.It is important to remove blunt foreign substances by endoscopy at an early stage.Simultaneously,it is imperative to enhance cooperation between dentistry and medicine and take appropriate measures

Immunofluorescent staining of Lm332 deposited by three cell lines.

<p>NHK (A, top), HSC-4 cells (A, center) and Lm332-HEK cells (A. bottom) were suspended in serum-free medium, inoculated at a cell density of 2×10³ cells/well on collagen-coated 8-well chamber slides and incubated for 6 h. The cultures were stained for F-actin with rhodamine phalloidin (left panels) and for Lm332 with the anti-α3 chain BG5 antibody and followed by a FITC-labeled secondary antibody (center panels), as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035546#s4" target="_blank">Materials and Methods</a>”. Right panels are merged images. In (B), Lm332-HEK cells were inoculated at a high cell density (1×10⁵ cells/well), incubated for 6 h (left panel) or 48 h (right panel), and stained for Lm332 as above.</p

Cell adhesion activity of Lm332-ECM and purified Lm332 toward NHK cells.

<p>ECMs from ß3γ2-HEK and Lm332-HEK cultures and Lm332-coated plates were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035546#pone-0035546-g004" target="_blank">Figure 4</a>. (A) Phase-contrast microscopic images of NHK cells after 10 min incubation. Original magnification, ×300. Lm332 protein was coated at 1.0 µg/ml on the plate. (B) NHK cells suspended in KGM growth medium were inoculated into each well and then incubated at 37°C for 10 min. After the incubation, non-adherent cells were removed, and adherent cells were quantified. Numerical values under three right columns indicate the concentration at µg/ml of coated Lm332 protein. Each bar represents the mean ± S.D. of the fluorescent intensity (FI) for adherent cells in triplicate assays. The data shown are representative of at least three independent experiments performed.</p

Fine structures of Lm332-ECM (left), ß3γ2-ECM (center) and purified Lm332 (right) were analyzed by TEM at a magnification of × 50,000.

<p>Bars indicate 100 nm.</p

Effect of sodium selenate on Lm332 deposition by NHK cells.

<p>(A) Immunoblotting analysis. NHK cells were inoculated in serum-free medium at a density of 4×10⁵ cells per 35-mm dish, incubated overnight, and treated with (+) or without (−) 0.1 mM sodium selenate (Sigma) at 37°C for 24 h. After the incubation, the ECM and CM were prepared from each culture and analyzed for the laminin α3 chain by immunoblotting as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035546#pone-0035546-g001" target="_blank">Figure 1</a>. (B) The ECMs from the control (none) and selenate-treated cultures (+selenate) were subjected to immunofluorescence staining with the anti-laminin α3 chain antibody BG5. (C) Phase-contrast micrographs of control and selenate-treated cultures. Other experimental conditions are described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035546#s4" target="_blank">Materials and Methods</a>”.</p

Differential detachment of NHK cells adhered to purified Lm332 and Lm332-ECM.

<p>NHK cells were inoculated onto the plates coated with 1 µg/ml purified Lm332 or deposited with Lm332-ECM and incubated for 1 h. After washing with PBS, the cells were incubated with trypsin/EDTA diluted 1∶35 in PBS (A) or with 10 mM EDTA alone (B). After incubation for the indicated lengths of time and then washing with PBS, the relative number of adherent cells was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035546#pone-0035546-g005" target="_blank">Figure 5</a>. Each bar indicates the mean ± S.D. of the fluorescent intensity (FI) for adherent cells in triplicate assays.</p

Interaction of purified Lm332 or Lm332-ECM with integrins.

<p>(A and B) To identify integrins responsible for cell adhesion, NHK cells suspended in the KGM medium were pretreated with the indicated integrin antibodies (2 µg/ml IgG) or EDTA for 30 min at room temperature and plated on the plates with purified Lm332 (A) or Lm332-ECM (B). After 20 min incubation, adherent cells were quantified. The relative number of adherent cells in the presence of control mouse or rat IgG was taken as 100%. Each bar represents the mean ± S.D. of the fluorescent intensity (FI) for adherent cells in triplicate assays. (C) Morphology of NHK cells was examined after incubation on Lm332-ECM in the presence of control mouse IgG, or the indicated anti-integrin neutral antibodies for 20 min. In the upper left culture (Lm332+IgG), NHK cells were incubated in a well precoated with 1.0 µg/ml purified Lm332 in the presence of the control IgG. Original magnification, ×300. The images shown are representative of at least three independent experiments performed. (D) Binding affinity of integrin α3ß1 to purified Lm332 and Lm332-ECM was determined by ELISA. Varied concentrations of purified integrin α3ß1 was allowed to bind to the plates coated with 1 µg/ml purified Lm332 (closed squares) or deposited with Lm332-ECM (open circles) in the presence of 1 mM MnCl₂. Bound integrin α3ß1 was quantified by ELISA using an anti-integrin α3ß1 polyclonal antibody. The amounts of integrin α3ß1 bound in the presence of 10 mM EDTA was taken as nonspecific binding and subtracted as the background. The results shown are the means of duplicate assays.</p