Rapid Adhesion Research Articles

Normal and malignant epithelial cells with stem-like properties have an extended G2 cell cycle phase that is associated with apoptotic resistance

BackgroundSubsets of cells with stem-like properties have been previously isolated from human epithelial cancers and their resistance to apoptosis-inducing stimuli has been related to carcinoma recurrence and treatment failure. The aim of this study was to investigate the mechanisms of resistance to apoptosis-inducing agents of cells with stem-like properties in both normal and malignant human epithelia.MethodsCells isolated from fresh human head and neck carcinomas (n = 11), cell lines derived from head and neck, prostate and breast human carcinomas (n = 7), and from normal human oral mucosa (n = 5), were exposed to various apoptosis-inducing stimuli (UV, Tumour Necrosis Factor, Cisplatin, Etoposide, and Neocarzinostatin). Flow cytometry for CD44 and epithelial-specific antigen (ESA) expression, colony morphology, tumour sphere formation and rapid adherence assays were used to identify the subset of cells with stem-like properties. Apoptosis, cell cycle and expression of various cell cycle checkpoint proteins were assessed (Western Blot, qPCR). The role of G2-checkpoint regulators Chk1 and Chk2 was investigated by use of debromohymenialdisine (DBH) and siRNA.ResultsIn both cancer biopsies and carcinoma cell lines a subset of CD44high cells showed increased clonogenicity, a significantly lower rate of apoptosis, and a significantly higher proportion of cells in the G2-phase of the cell cycle. An inverse correlation between the percentage of cells in G2-phase and the rate of apoptosis was found. Pulse-chase with iododeoxyuridine (IdU) demonstrated that CD44high carcinoma cells spent longer time in G2, even in un-treated controls. These cells expressed higher levels of G2 checkpoint proteins, and their release from G2 with BDH or Chk1 siRNA increased their rate of apoptosis. Low passage cultures of normal keratinocytes were also found to contain a subset of CD44high cells showing increased clonogenicity, and a similar pattern of G2-block associated with apoptotic resistance.ConclusionsThese data indicate that both normal and malignant human epithelial cells with stem-like properties show greater resistance to apoptosis associated with extended G2 cell cycle phase, and that this property is not a consequence of neoplastic transformation. Targeting G2 checkpoint proteins releases these cells from the G2-block and makes them more prone to apoptosis, implying an opportunity for improved therapeutic approaches.

Reactive Oxygen Species Level Defines Two Functionally Distinctive Stages of Inflammatory Dendritic Cell Development from Mouse Bone Marrow

Reactive oxygen species (ROS) have been implicated in various physiological activities. However, their role in dendritic cell (DC) activation and generation has not been investigated. Using the bone marrow-derived GM-CSF-induced ex vivo DC model, we characterize how induction of ROS correlates with inflammatory DC functionality and expansion. We describe that the functionality of GM-CSF-induced DCs is distinct in two developmental stages. Whereas division of DC-committed hematopoietic progenitor cells (HPCs) neared completion by day 6, the level of ROS soared after day 4. Day 3 ROS(lo) DCs were highly responsive to TLR stimuli such as LPS and zymosan by rapid upregulation of CD80, CD86, and MHC class II, in contrast to the low response of day 6 ROS(hi) DCs. ROS(hi) DCs could not initiate and sustain a significant level of NF-kappaB phosphorylation in response to LPS and zymosan, although demonstrating hyperactivation of p38 MAPK by LPS, in a fashion disparate to ROS(lo) DCs. ROS(lo) DCs stimulated a higher level of allogeneic and OVA-specific T cell proliferative responses, although ROS(hi) DCs were much more proficient in processing OVA. In response to pathogenic stimuli, ROS(hi) DCs also demonstrated rapid cellular adhesion and H(2)O(2) release, indicating their role in immediate microbial targeting. Moreover, HPC expansion and DC generation were dependent on the surge of ROS in an NADPH oxidase-independent manner. These findings point to the potential role of cellular ROS in mediating functionality and development of DCs from HPCs during inflammation.

The murine IL-8 homologues KC, MIP-2, and LIX are found in endothelial cytoplasmic granules but not in Weibel-Palade bodies

Rapid translocation of P-selectin from WPB to the surface of endothelial cells is crucial for early neutrophil recruitment to acute inflammatory lesions. Likewise, the chemokine CXCL8/IL-8 is sorted to WPB in human endothelial cells, but little is known about its functional importance in lack of a suitable animal model. Here, we explored the distribution of the functional IL-8 homologues CXCL1/KC, CXCL2/MIP-2, and CXCL5-6/LIX in resting and inflamed murine vessels by confocal microscopy and paired immunostaining with markers of WPB, discovering that these chemokines did not localize to WPB but displayed a granular pattern in a subset of vessels in healthy skin compatible with sorting to the type 2 endothelial compartment for regulated secretion. Moreover, all chemokines colocalized with VWF and P-selectin in platelets, suggesting that their storage in platelet alpha-granules might represent an alternative source of rapidly available, neutrophil-recruiting chemokines. In conclusion, WPB appear not to be involved in regulated secretion of chemokines in the mouse, and instead, the possible existence of type 2 granules and the role of platelets in rapid leukocyte adhesion deserve further attention.

Effects of homogeneous irradiation of electron beam with low potential on adhesive strength of polymethyl methacrylate composite sheet covered with nylon-6 film

By using homogeneous electron-beam (EB) irradiation, rapid adhesion between nylon-6 film and polymethyl methacrylate (PMMA) was successfully developed. Effects of homogeneous irradiation of electron beam with low potential (HIEBL) on adhesive strength of different polymers without glue were investigated. HIEBL less than 0.216MJ kg−1 (MGy) increased the adhesive strength and its strain of composites constructed with nylon-6 film and PMMA, although additional HIEBL at more than 0.432 MGy apparently decreased the adhesive strain. HIEBL less than 0.432 MGy also enhanced the elasticity (dσ/dε)o of composites. To evaluate the influence of HIEBL on the adhesive strength, electron spin resonance signals that relate to dangling bonds were observed. Because EB irradiation generated dangling bonds in polymethyl methacrylate and nylon-6, dangling bonds probably acted reactive and bonding sites to each polymer at interface. Therefore, HIEBL enhanced the adhesive strength as well as elasticity of the composites.

The Rho-family GEF Asef2 activates Rac to modulate adhesion and actin dynamics and thereby regulate cell migration

Asef2 is a recently identified Rho-family guanine nucleotide exchange factor (GEF) that has been implicated in the modulation of actin, but its function in cell migration and adhesion dynamics is not well understood. In this study, we show that Asef2 is an important regulator of cell migration and adhesion assembly and disassembly (turnover). Asef2 localizes with actin at the leading edge of cells. Knockdown of endogenous Asef2 impairs migration and significantly slows the turnover of adhesions. Asef2 enhances both Rac1 and Cdc42 activity in HT1080 cells, but only Rac1 is crucial for the Asef2-promoted increase in migration and adhesion turnover. Phosphoinositide 3-kinase (PI3K) and the serine/threonine kinase Akt are also essential for the Asef2-mediated effects on migration and adhesion turnover. Consistent with this, Asef2 increases the amount of active Akt at the leading edge of cells. Asef2 signaling leads to an overall decrease in Rho activity, which is crucial for stimulating migration and adhesion dynamics. Thus, our results reveal an important new role for Asef2 in promoting cell migration and rapid adhesion turnover by coordinately regulating the activities of Rho-family GTPases.

Isolation, culture and identification of epidermal stem cells from newborn mouse skin

In healthy individuals, skin integrity is maintained by epidermal stem cells which self-renew and generate daughter cells that undergo terminal differentiation. Epidermal stem cells represent a promising source of stem cells, and their culture has great potential in scientific research and clinical application. However, no single method has been universally adopted for identifying and isolating epidermal stem cells. Here, we reported the isolation and characterization of putative epidermal stem cells from newborn mouse skin. The keratinocytes were separated enzymatically. Putative epidermal stem cells were selected by rapid adherence on a composite matrix made of type I collagen and fibronectin. Unattached cells were discarded after 10 min, and the attached cells were cultured in a defined culture medium. The isolated cells showed the typical epidermal stem cell morphology. Immunofluorescence indicated that the cells were strongly stained for β1 integrin family of extracellular matrix receptors. In conclusion, mouse putative epidermal stem cells were successfully isolated from newborn mouse epidermis on the basis of high rapid adhesion to extracellular matrix proteins and cultured in vitro.

Dedifferentiation-derived cells exhibit phenotypic and functional characteristics of epidermal stem cells

Differentiated epidermal cells can dedifferentiate into stem cells or stem cell-like cells in vivo. In this study, we report the isolation and characterization of dedifferentiation-derived cells. Epidermal sheets eliminated of basal stem cells were transplanted onto the skin wounds in 47 nude athymic (BALB/c-nu/nu) mice. After 5 days, cells negative for CK10 but positive for CK19 and β1-integrin emerged at the wound-neighbouring side of the epidermal sheets. Furthermore, the percentages of CK19 and β1-integrin+ cells detected by flow cytometric analysis were increased after grafting (P < 0.01) and CK10+ cells in grafted sheets decreased (P < 0.01). Then we isolated these cells on the basis of rapid adhesion to type IV collagen and found that there were 4.56% adhering cells (dedifferentiation-derived cells) in the grafting group within 10 min. The in vitro phenotypic assays showed that the expressions of CK19, β1-integrin, Oct4 and Nanog in dedifferentiation-derived cells were remarkably higher than those in the control group (differentiated epidermal cells) (P < 0.01). In addition, the results of the functional investigation of dedifferentiation-derived cells demonstrated: (1) the numbers of colonies consisting of 5–10 cells and greater than 10 cells were increased 5.9-fold and 6.7-fold, respectively, as compared with that in the control (P < 0.01); (2) more cells were in S phase and G2/M phase of the cell cycle (proliferation index values were 21.02% in control group, 45.08% in group of dedifferentiation); (3) the total days of culture (28 days versus 130 days), the passage number of cells (3 passages versus 20 passages) and assumptive total cell output (1 × 105 cells versus 1 × 1012 cells) were all significantly increased and (4) dedifferentiation-derived cells, as well as epidermal stem cells, were capable of regenerating a skin equivalent, but differentiated epidermal cells could not. These results suggested that the characteristics of dedifferentiation-derived cells cultured in vitro were similar to epidermal stem cells. This study may also offer a new approach to yield epidermal stem cells for wound repair and regeneration.

Spontaneous Metastasis of Prostate Cancer Is Promoted by Excess Hyaluronan Synthesis and Processing

Accumulation of extracellular hyaluronan (HA) and its processing enzyme, the hyaluronidase Hyal1, predicts invasive, metastatic progression of human prostate cancer. To dissect the roles of hyaluronan synthases (HAS) and Hyal1 in tumorigenesis and metastasis, we selected nonmetastatic 22Rv1 prostate tumor cells that overexpress HAS2, HAS3, or Hyal1 individually, and compared these cells with co-transfectants expressing Hyal1 + HAS2 or Hyal1 + HAS3. Cells expressing only HAS were less tumorigenic than vector control transfectants on orthotopic injection into mice. In contrast, cells co-expressing Hyal1 + HAS2 or Hyal1 + HAS3 showed greater than sixfold and twofold increases in tumorigenesis, respectively. Fluorescence and histological quantification revealed spontaneous lymph node metastasis in all Hyal1 transfectant-implanted mice, and node burden increased an additional twofold when Hyal1 and HAS were co-expressed. Cells only expressing HAS were not metastatic. Thus, excess HA synthesis and processing in concert accelerate the acquisition of a metastatic phenotype by prostate tumor cells. Intratumoral vascularity did not correlate with either tumor size or metastatic potential. Analysis of cell cycle progression revealed shortened doubling times of Hyal1-expressing cells. Both adhesion and motility on extracellular matrix were diminished in HA-overproducing cells; however, motility was increased twofold by Hyal1 expression and fourfold to sixfold by Hyal1/HAS co-expression, in close agreement with observed metastatic potential. This is the first comprehensive examination of these enzymes in a relevant prostate cancer microenvironment.

Transmitting tissue architecture in basal‐relictual angiosperms: Implications for transmitting tissue origins

Carpel transmitting tissue is a major floral innovation that is essential for angiosperm success. It facilitates the rapid adhesion, hydration, and growth of the male gametophyte to the female gametophyte. As well, it functions as a molecular screen to promote male gametophytic competition and species-specific recognition and compatibility. Here, we characterize the transmitting tissue extracellular matrix (ECM) and pollen tube growth in basal-relictual angiosperms and test the hypothesis that a freely flowing ECM (wet stigma) was ancestral to a cuticle-bound ECM (dry stigma). We demonstrate that the most recent common ancestor of extant angiosperms produced an ECM that was structurally and functionally equivalent to a dry stigma. Dry stigmas are composed of a cuticle and primary wall that contains compounds that facilitate the adhesion and growth of the male gametophyte. These compounds include methyl-esterified homogalacturonans, arabinogalactan-proteins, and lipids. We propose that transmitting tissue evolved in concert with an increase in cuticle permeability that resulted from modifications in the biosynthesis and secretion of fatty acids needed for cuticle construction. Increased cuticle permeability exposed the male gametophyte to pre-existing molecules that enabled rapid male gametophyte adhesion, hydration, and growth as well as species-specific recognition and compatibility.

A highly phagocytic cell line TO from Atlantic salmon is CD83 positive and M-CSFR negative, indicating a dendritic-like cell type

Leucocyte cell lines are valuable tools for immunological studies. In this study the TO cell line, originating from Atlantic salmon head kidney leucocytes, is described with respect to enzyme cytochemistry, functional studies, reactivity with leucocyte specific antibodies and immune gene expression. Pronounced characteristics of the TO cell line are the rapid adherence to the plastic growth surface, high phagocytic capacity and bactericidal functions. No respiratory burst activity, and little or no NO production were detected under the experimental conditions tested, and thus the TO cells appear to have other effective killing mechanisms. The cells are reactive with a leucocyte specific monoclonal antibody (MAb), but does not bind a neutrophil specific MAb or stain for myeloperoxidase. Real-time RT-PCR showed the expression in TO cells of several immune genes, some of which were significantly regulated following LPS stimulation. The expression of CD83 might indicate a dendritic cell (DC) origin of the TO cells, as this marker is considered a hallmark for DC. Expression of TCR-α or the macrophage marker M-CSFR was not detected. Based on the present analyses the TO cells display a mixture of known characteristics for macrophages and DCs. At the same time the TO cells lack some central functions of phagocytic/myeloid cells. As the TO cells are developed to a long-term culture one cannot exclude that some functions might have been lost in this process. Nevertheless, the features of the TO cells indicate their potential as a model system for immunological studies of salmon phagocytic cells.

Limbal microenvironment can induce transdifferentiation of hair follicle stem cells into corneal epithelial‐like cells

Abstract Purpose To investigate the plasticity of murine vibrissa hair follicle (HF) stem cells regarding differentiation into corneal epithelial‐like cells through modulation by limbus‐specific microenvironmental factors. Methods HFs were isolated and the dissociated bulge stem cells enriched by clonal expansion, and subcultivated on various extracellular matrices (collagen type IV, laminin‐1, laminin‐5, fibronectin) in DMEM/F12 medium supplemented with different conditioned media (CMs). CMs were derived from central and peripheral corneal fibroblasts and limbal stromal fibroblasts. Growth potential and cellular phenotype were evaluated by light and electron microscopy, real‐time‐PCR and immunohistochemistry using antibodies against stem cell (CK15, integrin α6) and differentiation markers (CK12) or (CK10). Results Laminin‐5 and collagen IV promoted rapid cell adhesion, proliferation, and generation of confluent, regularly arranged epitheloid cell sheets, whereas laminin‐1 and fibronectin adversely affected these cell properties. Addition of CMs differentially influenced cellular phenotype and differentiation. Limbal fibroblast CM induced differentiation of cuboid cells showing ultrastructural characteristics of a corneal epithelial phenotype. Moreover, it markedly increased the number of CK12 immunopositive cells while decreasing expression of CK10 opposed to the other CMs. Conclusion Hair follicle stem cells are capable of differentiating into corneal epithelial‐like cells in vitro when exposed to a limbus‐specific microenvironment. Therefore, the HF may be an alternative therapeutic source of multipotent stem cells for generation of autologous epithelial cell sheets for ocular surface reconstruction.

Rapid adhesion and proliferation of keratinocytes on the gold colloid/chitosan film scaffold

The gold colloid/chitosan film scaffold, which could enhance the attached ratio and accelerate proliferation of newborn mice keratinocytes, was fabricated by nanotechnology and self-assembly technology. This nanometer scaffold was characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The keratinocytes were cultured and observed on three different extracellular matrices (ECM): gold colloid/chitosan film scaffold, chitosan film and cell culture plastic (control groups). 6 h, 12 h, 24 h after inoculation, the cell attached ratios were calculated respectively. In comparison to control groups, this scaffold could significantly ( P < 0.01) increase the attached ratio of keratinocytes and promote their growth. Meanwhile, there were not any fusiform fibroblasts growing on this scaffold. The rapidly proliferating keratinocytes were indentified and characterized by immunohistochemistry and transmissive electron microscope (TEM), which showed the cells maintain their biological activity well. The results indicated that gold colloid/chitosan film scaffold was nontoxic to keratinocytes, and was a good candidate for wound dressing in skin tissue engineering.

Application of microwave heat to paraffin sections: rapid adhesion of sections to slides

(2008). Application of microwave heat to paraffin sections: rapid adhesion of sections to slides. British Journal of Biomedical Science: Vol. 65, No. 4, pp. 200-201.

Eradication of B Cell Malignancies by Anti-3rd Party CTLs: A Novel Role for MHC Class I.

Induction of donor type chimerism under reduced intensity conditioning in the absence of GVHD represents a major challenge in transplantation biology. Despite their remarkable potent veto activity, CD8 CTLs could not be used to enhance engraftment due to their GVH reactivity. To address this problem we developed a new approach for the generation of human host-nonreactive CTLs, based on stimulation of donor CD8 T cells against 3rd party stimulators under IL-2 deprivation. A major attribute of such anti-3rd party CTLs reported recently by our group is their ability to eradicate pathological B-CLL cells. This B-CLL killing was shown to be independent of TCR recognition and it was found to be mediated both by autologous and by allogeneic anti-3rd party CTLs. In the present study we demonstrate that anti-3rd party CTLs can also efficiently eradicate primary mantle lymphoma cells and Burkitt and mantle cell-lines. Furthermore, the use of lymphoma cell lines has enabled to define several aspects of the mechanism leading to their apoptosis. Thus, we found that anti-3rd party CTLs avidly bind to target lymphoma cells and form stable aggregates within the first minutes of contact. Conjugate formation as well as the actual killing of the tumor cells were effectively inhibited by blocking antibodies against LFA1 or ICAM1 but not against ICAM3. Moreover, effective inhibition was attained only when applying anti-LFA1 to the CTLs and anti-ICAM1 to the tumor cells (100%±10% and 72%±7% inhibition, respectively) but not vice-versa. Given the role of CD8-MHC class I interaction in veto mechanisms, we sought to evaluate its role in the killing of lymphoma cells by human anti-3rd party CTLs. Indeed, in 10 experiments survival of target lymphoma cells was increased from 50%±5% to 82%±1% upon addition of anti HLA-ABC Ab to the incubation with anti 3rd party CTLs. A similar survival increase to 77.5%±1% was found upon the addition of anti-CD8 Ab, suggesting that CD8 binding to MHC class I constant region is required for lymphoma cell killing. The important role of MHC class I was further established by comparing CTL-mediated killing of Daudi lymphoma cells lacking cell surface MHC class I expression and Daudi cells with reconstituted surface MHC class I due to stable β2-microglobulin overexpression (β2m-Daudi). Thus in contrast to the poor killing of Daudi cells which did not exceed 20%, the β2m-Daudi cell killing was comparable to that exhibited with burkitt lymphoma line expressing MHC class1 (56%±1% and 63%±3%, respectively in 4 experiments). Importantly, FACS analysis showed that, MHC I expression did not influence conjugate formation, but was rather involved in the induction of apoptosis (annexin V). Furthermore, a similar pattern was also found in vivo when β2m-Daudi or Daudi cells were infused into the peritoneum of SCID mice in the presence or absence of a 2.5-fold excess of anti 3rd party CTLs. In 3 experiments, FACS analysis of the cells recovered from the peritoneum showed that the CTLs killed 91% of the β2m-Daudi cells as opposed to 13% killing of the Daudi cells. Taken together, our results suggest that TCR independent killing of B cell malignancies by anti-3rd party CTLs is mediated via a rapid adhesion through ICAM1-LFA1 binding, followed by slow induction of apoptosis upon a critical interaction between CD8 on the CTL and MHC class I on the tumor cell.

Molecular Interactions between T Cells and Fibroblast-Like Synoviocytes: Role of Membrane Tumor Necrosis Factor-α on Cytokine-Activated T Cells

The mechanism of fibroblast-like synoviocyte (FLS) transformation into an inflammatory phenotype in rheumatoid arthritis (RA) is not fully understood. FLS interactions with invading leukocytes, particularly T cells, are thought to be a critical component of this pathological process. Resting T cells and T cells activated through the T-cell receptor have previously been shown to induce inflammatory cytokine production by FLS. More recently, a distinct population of T cells has been identified in RA synovium that phenotypically resembles cytokine-activated T (Tck) cells. Using time lapse microscopy, the interactions of resting, superantigen-activated, and cytokine-activated T cells with FLS were visualized. Rapid and robust adhesion of Tck and superantigen-activated T cells to FLS was observed that resulted in flattening of the T cells and a crawling movement on the FLS surface. Tck also readily activated FLS to produce interleukin IL-6 and IL-8 in a cell contact-dependent manner that was enhanced by exogenous IL-17. Although LFA-1 and ICAM-1 co-localized at the Tck-FLS synapse, blocking the LFA-1/ICAM-1 interaction did not substantially inhibit Tck effector function. However, antibody blocking of membrane tumor necrosis factor (TNF)-alpha on the Tck surface did inhibit FLS cytokine production, thus illustrating a novel mechanism for involvement of TNF-alpha in cell-cell interactions in RA synovium and for the effectiveness of TNF-alpha blockade in the treatment of RA.

Rapid Adhesion to Collagen Isolates Murine Keratinocytes with Limited Long-Term Repopulating Ability In Vivo Despite High Clonogenicity In Vitro

A prevalent belief in epidermal biology is that stem cells are highly clonogenic; that is, they have the ability to produce many large colonies in vitro. However, it has been well-established in hematology, and recently suggested in epithelial biology, that short-term in vitro clonogenic assays may not be reliable predictors of long-term in vivo repopulating ability. Numerous groups have shown that rapid adhesion to collagen selects for highly clonogenic keratinocytes, but it has not been demonstrated whether this subpopulation is enriched in stem cells as defined by long-term repopulating ability in vivo. We found that although rapid adhesion to collagen (within 5 minutes) selected for cells with increased short-term colony forming ability in vitro, these cells were not enriched in long-term proliferative ability in vitro or in repopulating ability in vivo after 9 weeks. Conversely, keratinocytes that did not adhere to collagen (after 20 minutes) were less clonogenic in short-term assays but possessed equivalent long-term proliferative ability in vitro and superior long-term repopulating ability in vivo. Both the rapidly adherent cell and not rapidly adherent cell populations contained small, noncomplex basaloid cells, expressed integrin alpha2 (a collagen IV receptor), and expressed the putative epidermal stem cell phenotype integrin alpha6(hi)CD71(lo). Our results indicate that the superior short-term colony forming ability of collagen-adherent murine keratinocytes does not correlate with long-term repopulating ability in vitro or in vivo and that proliferation in vitro is not a reliable surrogate for stem cell behavior in vivo.

A novel peptide sequence in perlecan domain IV supports cell adhesion, spreading and FAK activation

Perlecan/HSPG2 is a large, multi-domain, multifunctional heparan sulfate proteoglycan with a wide tissue distribution. With the exception of its unique domain I, each of perlecan's other four domains shares sequence similarity to other protein families including low density lipoprotein (LDL) receptor, laminin alpha chain, neural cell adhesion molecule (NCAM), immunoglobulin (Ig) superfamily members, and epidermal growth factor (EGF). Previous studies demonstrated that glycosaminoglycan-bearing perlecan domain I supports early chondrogenesis and growth factor delivery. Other sites in the core protein interact with other matrix molecules and support cell adhesion, although the peptide sequences involved remain unidentified. To identify novel functional motifs within perlecan, we used a bioinformatics approach to predict regions likely to be on the exterior of the folded protein. Unique hydrophilic sequences of about 18 amino acids were selected for testing in cell adhesion assays. A novel peptide sequence (TWSKVGGHLRPGIVQSG) from an immunoglobulin (Ig) repeat in domain IV supported rapid cell adhesion, spreading and focal adhesion kinase (FAK) activation when compared to other peptides, a randomly scrambled sequence of the domain IV peptide or a negative control protein. MG-63 human osteosarcoma cells, epithelial cells and multipotent C 3H10T1/2 cells, but not bone marrow cells, rapidly, i.e., within 30 min, formed focal adhesions and assembled an actin cytoskeleton on domain IV peptide. Cell lines differentially adhered to the domain IV peptide, suggesting adhesion is receptor specific. Adhesion was divalent cation independent and heparin sensitive, a finding that may explain some previously poorly understood observations obtained with intact perlecan. Collectively, these studies demonstrate the feasibility of using bioinformatics-based strategies to identify novel functional motifs in matrix proteins such as perlecan.

Structural features and functional domains of amassin-1, a cell-binding olfactomedin protein

Amassin-1 mediates a rapid cell adhesion that tightly adheres sea urchin coelomocytes (body cavity immunocytes) together. Three major structural regions exist in amassin-1: a short beta region, 3 coiled coils, and an olfactomedin domain. Amassin-1 contains 8 disulfide-bonded cysteines that, upon reduction, render it inactive. Truncated forms of recombinant amassin-1 were expressed and purified from Pichia pastoris and their disulfide bonding and biological activities investigated. Expressed alone, the olfactomedin domain contained 2 intramolecular disulfide bonds, existed in a monomeric state, and inhibited amassin-1-mediated clotting of coelomocytes by a calcium-dependent cell-binding activity. The N-terminal beta region, containing 3 cysteines, was not required for clotting activity. The coiled coils may dimerize amassin-1 in a parallel orientation through a homodimerizing disulfide bond. Neither amassin-1 fragments that were disulfide-linked as dimers or that were engineered to exist as dimers induced coelomocytes clotting. Clotting required higher multimeric states of amassin-1, possibly tetramers, which occurred through the N-terminal beta region and (or) the first segment of coiled coils.

Rapid adhesion of Stagonospora nodorum spores to a hydrophobic surface requires pre-formed cell surface glycoproteins

Adhesion of fungal pathogens to leaf surfaces is an important first step in the infection process. Previous work on Stagonospora nodorum, a major necrotrophic pathogen of wheat and other cereals, has shown that conidia attach rapidly to a hydrophobic surface and this is followed by the active secretion of extracellular matrix material to consolidate adhesion. In this paper the role of pre-formed spore surface glycoproteins in the rapid adhesion of S. nodorum conidia to an artificial surface, polystyrene, has been investigated. Sodium dodecyl sulphate (SDS) and the enzymes chitinase and lyticase have been used to release cell wall glycoproteins from spores and these have been identified using SDS polyacrylamide gel electrophoresis (PAGE) and Western blotting. Labelling with fluorescently tagged lectins has also been used to study the spore surface. The results show that there are a small number of glycoproteins non-covalently and covalently attached to other components in the spore wall, which is not a uniform structure. The effects of proteases, lectins, and other treatments of spores in an adhesion assay have been used to show that pre-formed glycoproteins are involved in rapid adhesion to a hydrophobic surface. There is also evidence for a rapid release of glycoproteins by spores that is also involved in adhesion and this is not an active process.

Tight junction formation in early Xenopus laevis embryos: identification and ultrastructural characterization of junctional crests and junctional vesicles

How tight junctions (TJ) form during early amphibian embryogenesis is still an open question. We used time-lapse video microscopy, scanning electron microscopy (SEM), TEM and freeze-fracture to gain new insight into TJ biogenesis in early clevages of Xenopus laevis. Video analysis suggests three phases in junction formation between blastomeres. A first "waiting" phase, where new unpigmented lateral membranes are generated. A second "mixing" phase, where the unpigmented lateral membrane is separated from the pigmented apical membrane by an area showing a limited degree of intermingling of cortical pigment. And a third "sealing" phase, characterized by the formation of cingulin-containing boundaries between membrane domains, and their rapid directional adhesion in a zipper-like fashion. By SEM, we characterized these boundaries ("junctional crests", JC) as arrays of villiform protrusions at the border between old and new membranes. In the 2-cell embryo, JC are deeply located, and thus not visible at the surface, but they become increasingly more superficial as cleavages progress. After adjacent blastomeres have adhered to each other, fractured JC display linear arrays of junctional vesicles (JV) of 1-3 mum diameter. TEM analysis shows that JV are symmetrically located near the apposed membranes of adjacent blastomeres, and that the membranes near the JV display focal sites of intimate contact, typical of TJ. Freeze-fracture analysis confirms that intramembrane fibrils, typical of TJ, are present at adhesion sites. We conclude that TJ are formed following the sealing of JC, through the recruitment, sorting and assembly of membrane and cytoplasmic proteins at or near JV.