Source of Energetic Protons in the 2014 September 1 Sustained Gamma-ray Emission Event.

We report on the source of > 300 MeV protons during the SOL2014-09-01 sustained gamma-ray emission (SGRE) event based on multi-wavelength data from a wide array of space- and ground-based instruments. Based on the eruption geometry we provide concrete explanation for the spatially and temporally extended γ -ray emission from the eruption. We show that the associated flux rope is of low inclination (roughly oriented in the east-west direction), which enables the associated shock to extend to the frontside. We compare the centroid of the SGRE source with the location of the flux rope's leg to infer that the high-energy protons must be precipitating between the flux rope leg and the shock front. The durations of the SOL2014-09-01 SGRE event and the type II radio burst agree with the linear relationship between these parameters obtained for other SGRE events with duration ≥ 3 hrs . The fluence spectrum of the SEP event is very hard, indicating the presence of high-energy (GeV) particles in this event. This is further confirmed by the presence of an energetic coronal mass ejection with a speed > 2000 km s - 1 , similar to those in ground level enhancement (GLE) events. The type II radio burst had emission components from metric to kilometric wavelengths as in events associated with GLE events. All these factors indicate that the high-energy particles from the shock were in sufficient numbers needed for the production of γ -rays via neutral pion decay.

Long-term Solar Activity Studies using Microwave Imaging Observations and Prediction for Cycle 25.

We use microwave imaging observations from the Nobeyama Radioheliograph at 17 GHz for long-term studies of solar activity. In particular, we use the polar and low-latitude brightness temperatures as proxies to the polar magnetic field and the active-regions, respectively. We also use the location of prominence eruptions as a proxy to the filament locations as a function of time. We show that the polar microwave brightness temperature is highly correlated with the polar magnetic field strength and the fast solar wind speed. We also show that the polar microwave brightness at one cycle is correlated with the low latitude brightness with a lag of about half a solar cycle. We use this correlation to predict the strength of the solar cycle: the smoothed sunspot numbers in the southern and northern hemispheres can be predicted as 89 and 59, respectively. These values indicate that cycle 25 will not be too different from cycle 24 in its strength. We also combined the rush to the pole data from Nobeyama prominences with historical data going back to 1860 to study the north-south asymmetry of sign reversal at solar poles. We find that the reversal asymmetry has a quasi-periodicity of 3-5 cycles.

Basic research and clinical application of beta-tricalcium phosphate (ß-TCP).

The mechanism of bone substitute resorption involves two processes: solution-mediated and cell-mediated disintegration. In our previous animal studies, the main resorption process of beta-tricalcium phosphate (ß-TCP) was considered to be cell-mediated disintegration by TRAP-positive cells. Thus, osteoclast-mediated resorption of ß-TCP is important for enabling bone formation. We also report the results of treatment with ß-TCP graft in patients since 1989. Two to three weeks after implantation, resorption of ß-TCP occurred from the periphery, and then continued toward the center over time. Complete or nearly complete bone healing was achieved in most cases within a few years and was dependent upon the amount of implanted material, the patient's age, and the type of bone (cortical or cancellous). We have previously reported that an injectable complex of ß-TCP granules and collagen supplemented with rhFGF-2 enabled cortical bone regeneration of rabbit tibiae. Based on the experimental results, we applied this technique to the patients with femoral and humeral fractures in elderly patients, and obtained bone union.

Measurement and comparison of individual external doses of high-school students living in Japan, France, Poland and Belarus-the 'D-shuttle' project.

Twelve high schools in Japan (of which six are in Fukushima Prefecture), four in France, eight in Poland and two in Belarus cooperated in the measurement and comparison of individual external doses in 2014. In total 216 high-school students and teachers participated in the study. Each participant wore an electronic personal dosimeter 'D-shuttle' for two weeks, and kept a journal of his/her whereabouts and activities. The distributions of annual external doses estimated for each region overlap with each other, demonstrating that the personal external individual doses in locations where residence is currently allowed in Fukushima Prefecture and in Belarus are well within the range of estimated annual doses due to the terrestrial background radiation level of other regions/countries.

Antiproliferative activity of ecteinascidin 743 is dependent upon transcription-coupled nucleotide-excision repair.

While investigating the novel anticancer drug ecteinascidin 743 (Et743), a natural marine product isolated from the Caribbean sea squirt, we discovered a new cell-killing mechanism mediated by DNA nucleotide excision repair (NER). A cancer cell line selected for resistance to Et743 had chromosome alterations in a region that included the gene implicated in the hereditary disease xeroderma pigmentosum (XPG, also known as Ercc5). Complementation with wild-type XPG restored the drug sensitivity. Xeroderma pigmentosum cells deficient in the NER genes XPG, XPA, XPD or XPF were resistant to Et743, and sensitivity was restored by complementation with wild-type genes. Moreover, studies of cells deficient in XPC or in the genes implicated in Cockayne syndrome (CSA and CSB) indicated that the drug sensitivity is specifically dependent on the transcription-coupled pathway of NER. We found that Et743 interacts with the transcription-coupled NER machinery to induce lethal DNA strand breaks.

CTAR®system, a hypoxia-induced and targeted anti-aging and repair system on human skin.

OBJECTIVE: The effective protection of skin cells and timely repair of damaged ones are of great significance for the maintenance of human skin's normal functions. Researches on skin cells mainly focus on the functions of repair factors. Relatively few studies have been made to clarify how skin cells sense the environment, absorb and transport nutrients, and achieve self-healing. CTAR®system, a new system, is proposed and demonstrated against human skin aging. MATERIALS AND METHODS: This study is made based on the research outcomes of the 2013 and 2019 Nobel Prize in Physiology or Medicine: precise regulation of cellular vesicle transport networks, and cellular perception and responding mechanisms to oxygen. The technologies and methods derived from the award-winning theories were applied in the field of skin anti-aging. RESULTS: It has shown that skin aging is a result of a series of injuries that have not been adequately repaired and then accumulate. The latest research on anti-aging of skin including channel identification, vesicle transport, induced activation, and damage repair has spawned CTAR®system, a hypoxia-induced and targeted anti-aging and repair system. CONCLUSIONS: We determined that a considerable part of skin aging problems can be partially solved by taking full advantage of the vesicular trafficking system in skin and improving skin's metabolism levels and physiological functions under low oxygen levels with CTAR®system.

Human neutrophil adherence to laminin in vitro. Evidence for a distinct neutrophil integrin receptor for laminin.

We used mAbs against polymorphonuclear leukocyte (PMN) surface proteins to investigate the mechanisms by which stimulated human neutrophils (PMNs) adhere in vitro to laminin, the major glycoprotein of mammalian basement membrane. mAb IB4, which is directed against the common beta 2 chain of the CD11/CD18, only partially inhibited the adherence of PMA-stimulated PMNs to both laminin and to subendothelial matrices. In contrast, IB4 completely inhibited PMA-stimulated PMN adherence to gelatin, fibronectin, collagen IV, and endothelial cell monolayers. PMA-stimulated PMNs from a patient with severe congenital CD11/CD18 deficiency also adhered to laminin, but not to gelatin or endothelial cell monolayers. Therefore, PMA-stimulated PMNs adhere to laminin by both CD11/CD18-dependent and CD11/CD18-independent mechanisms. Expression of CD11/CD18-independent adherence to laminin was agonist dependent, occurring after stimulation with the calcium ionophore A23187 and recombinant TNF-alpha, but not with the chemotactic factors FMLP, platelet activating factor, or recombinant human C5a. Expression of CD11/CD18-independent adherence was also divalent cation dependent, occurring in the presence of Mg2+ but not Ca2+ as the sole added divalent cation. The mAbs AIIB2 and 13, which are directed against the beta 1 subunit of the VLA integrins, significantly inhibited the CD11/CD18-independent adherence of normal PMNs to laminin, and completely abolished the adherence of CD11/CD18-deficient PMNs to laminin. Both anti-beta 1 mAbs bound to PMNs, as demonstrated by flow cytometry, and immunoprecipitated a membrane molecule of Mr 130,000 daltons from 125I-labeled, detergent-solubilized PMNs. These data suggest that human PMNs possess beta 1 and beta 2 classes of integrins, and that both mediate PMN adherence.

Activation of CD4 cells by fibronectin and anti-CD3 antibody. A synergistic effect mediated by the VLA-5 fibronectin receptor complex.

In this study, fibronectin synergized with anti-CD3 antibody to promote CD4 cell proliferation in a serum-free culture system. The cell-adhesive domain plus additional regions of the fibronectin molecule are involved in this synergy. Anti4B4(CDw29) antibody blocked the activation of CD4 cells in this system. Furthermore, it is the VLA-5 protein within the set of molecules recognized by anti-4B4 that serves as a fibronectin receptor on the CD4 lymphocytes. The VLA-5 fibronectin receptor was mainly expressed on CD4+ CD45R-CDw29+ cells and may in part contribute to the unique function of these cells.

Characterization of a 140-kD avian cell surface antigen as a fibronectin-binding molecule.

A 140,000-D protein cell surface antigen (140k) complex has been implicated in fibronectin-mediated cell-substratum attachment. We have used three different experimental systems to evaluate the hypothesis that this 140k complex can function as a fibronectin receptor. A monoclonal antibody that binds to the 140k complex specifically inhibits the direct binding of 3H-labeled 75,000-D fibronectin cell-binding fragment (f75k) to chicken embryo fibroblasts in suspension. The 140k complex is retarded in its passage through an affinity column consisting of immobilized f75k, and this interaction is specifically inhibited by a synthetic peptide that contains the fibronectin cell-recognition signal sequence. Finally, exogenous purified 140k complex inhibits the attachment and spreading of chicken embryo fibroblasts on fibronectin-coated substrates. Thus, our results indicate that the 140k complex can bind directly to fibronectin and is likely to be a fibronectin receptor for chicken cells.

Regulation of fibronectin receptor distribution.

To determine the role of each intracellular domain of the fibronectin receptor in receptor distribution, chimeric receptors were constructed containing the human interleukin-2 receptor (gp55 subunit) as the extracellular and transmembrane domains, in combination with either the alpha 5 or beta 1 intracellular domain of the fibronectin receptor as the cytoplasmic domain. These chimeric receptors were transiently expressed in normal fibroblasts, and their localization on the cell surface was determined by immunofluorescence using antibodies to the human interleukin-2 receptor. The alpha 5 chimera was expressed diffusely on the plasma membrane. The beta 1 chimera, however, colocalized with the endogenous fibronectin receptor at focal contacts of cells spread on fibronectin. On cells spread in the presence of serum, the beta 1 chimera colocalized both with the fibronectin receptor at sites of extracellular fibronectin fibrils and with the vitronectin receptor at focal contacts. The beta 1 intracellular domain alone, therefore, contains sufficient information to target the chimeric receptor to regions of the cell where ligand-occupied integrin receptors are concentrated. The finding that the beta 1 chimeric protein behaves like a ligand-occupied receptor, even though the beta 1 chimera cannot itself bind extracellular ligand, suggests an intracellular difference between occupied and unoccupied receptors, and predicts that the distribution of integrin receptors can be regulated by ligand occupancy. We tested this prediction by providing a soluble cell-binding fragment of fibronectin to cells spread on laminin. Under conditions preventing further ligand adsorption to the substrate, this treatment nevertheless resulted in the relocation of diffuse fibronectin receptors to focal contacts. Similarly, a redistribution of diffuse vitronectin receptors to focal contacts occurred on cells spread on laminin after the addition of the small soluble peptide GRGDS. We conclude that the propensity for receptor redistribution to focal contacts driven by the beta 1 cytoplasmic domain alone is suppressed in heterodimeric unoccupied fibronectin receptors, and that ligand occupancy can release this constraint. This redistribution of integrin receptors after the binding of a soluble substrate molecule may provide a direct means of assembling adhesion sites.

Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion.

We have compared the molecular specificities of the adhesive interactions of melanoma and fibroblastic cells with fibronectin. Several striking differences were found in the sensitivity of the two cell types to inhibition by a series of synthetic peptides modeled on the Arg-Gly-Asp-Ser (RGDS) tetrapeptide adhesion signal. Further evidence for differences between the melanoma and fibroblastic cell adhesion systems was obtained by examining adhesion to proteolytic fragments of fibronectin. Fibroblastic BHK cells spread readily on fl3, a 75-kD fragment representing the RGDS-containing, "cell-binding" domain of fibronectin, but B16-F10 melanoma cells could not. The melanoma cells were able to spread instead on f9, a 113-kD fragment derived from the large subunit of fibronectin that contains at least part of the type III connecting segment difference region (or "V" region); f7, a fragment from the small fibronectin subunit that lacks this alternatively spliced polypeptide was inactive. Monoclonal antibody and fl3 inhibition experiments confirmed the inability of the melanoma cells to use the RGDS sequence; neither molecule affected melanoma cell spreading, but both completely abrogated fibroblast adhesion. By systematic analysis of a series of six overlapping synthetic peptides spanning the entire type III connecting segment, a novel attachment site was identified in a peptide near the COOH-terminus of this region. The tetrapeptide sequence Arg-Glu-Asp-Val (REDV), which is somewhat related to RGDS, was present in this peptide in a highly hydrophilic region of the type III connecting segment. REDV appeared to be functionally important, since this synthetic tetrapeptide was inhibitory for melanoma cell adhesion to fibronectin but was inactive for fibroblastic cell adhesion. REDV therefore represents a novel adhesive recognition signal in fibronectin that possesses cell type specificity. These results suggest that, for some cell types, regulation of the adhesion-promoting activity of fibronectin may occur by alternative mRNA splicing.

Inhibition of binding of fibronectin to matrix assembly sites by anti-integrin (alpha 5 beta 1) antibodies.

Fibroblasts have cell surface sites that mediate assembly of plasma and cellular fibronectin into the extracellular matrix. Cell adhesion to fibronectin can be mediated by the interaction of an integrin (alpha 5 beta 1) with the Arg-Gly-Asp-Ser (RGDS)-containing cell adhesion region of fibronectin. We have attempted to elucidate the role of the alpha 5 beta 1 fibronectin receptor in assembly of fibronectin in matrices. Rat monoclonal antibody mAb 13, which recognizes the integrin beta 1 subunit, completely blocked binding and matrix assembly of 125I-fibronectin as well as binding of the 125I-70-kD amino-terminal fragment of fibronectin (70 kD) to fibroblast cell layers. Fab fragments of the anti-beta 1 antibody were also inhibitory. Antibody mAb 16, which recognizes the integrin alpha 5 subunit, partially blocked binding of 125I-fibronectin and 125I-70-kD. When cell layers were coincubated with fluoresceinated fibronectin and either anti-beta 1 or anti-alpha 5, anti-beta 1 was a more effective inhibitor than anti-alpha 5 of binding of labeled fibronectin to the cell layer. Inhibition of 125I-fibronectin binding by anti-beta 1 IgG occurred within 20 min. Inhibition of 125I-fibronectin binding by anti-beta 1 Fab fragments or IgG could not be overcome with increasing concentrations of fibronectin, suggesting that anti-beta 1 and exogenous fibronectin may not compete for the same binding site. No beta 1-containing integrin bound to immobilized 70 kD. These data indicate that the beta 1 subunit plays an important role in binding and assembly of exogenous fibronectin, perhaps by participation in the organization, regeneration, or cycling of the assembly site rather than by a direct interaction with fibronectin.

Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization.

We have developed two rat mAbs that recognize different subunits of the human fibroblast fibronectin receptor complex and have used them to probe the function of this cell surface heterodimer. mAb 13 recognizes the integrin class 1 beta polypeptide and mAb 16 recognizes the fibronectin receptor alpha polypeptide. We tested these mAbs for their inhibitory activities in cell adhesion, spreading, migration, and matrix assembly assays using WI38 human lung fibroblasts. mAb 13 inhibited the initial attachment as well as the spreading of WI38 cells on fibronectin and laminin substrates but not on vitronectin. Laminin-mediated adhesion was particularly sensitive to mAb 13. In contrast, mAb 16 inhibited initial cell attachment to fibronectin substrates but had no effect on attachment to either laminin or vitronectin substrates. When coated on plastic, both mAbs promoted WI38 cell spreading. However, mAb 13 (but not mAb 16) inhibited the radial outgrowth of cells from an explant on fibronectin substrates. mAb 16 also did not inhibit the motility of individual fibroblasts on fibronectin in low density culture and, in fact, substantially accelerated migration rates. In assays of the assembly of an extracellular fibronectin matrix by WI38 fibroblasts, both mAbs produced substantial inhibition in a concentration-dependent manner. The inhibition of matrix assembly resulted from impaired retention of fibronectin on the cell surface. Treatment of cells with mAb 16 also resulted in a striking redistribution of cell surface fibronectin receptors from a streak-like pattern to a relatively diffuse distribution. Concomitant morphological changes included decreases in thick microfilament bundle formation and reduced adhesive contacts of the streak-like and focal contact type. Our results indicate that the fibroblast fibronectin receptor (a) functions in initial fibroblast attachment and in certain types of adhesive contact, but not in the later steps of cell spreading; (b) is not required for fibroblast motility but instead retards migration; and (c) is critically involved in fibronectin retention and matrix assembly. These findings suggest a central role for the fibronectin receptor in regulating cell adhesion and migration.

Neurite extension of chicken peripheral nervous system neurons on fibronectin: relative importance of specific adhesion sites in the central cell-binding domain and the alternatively spliced type III connecting segment.

Fibronectin contains at least two domains that support cell adhesion. One is the central cell-binding domain that is recognized by a variety of cell types, including fibroblasts. The second, originally identified by its ability to support melanoma cell adhesion, is located in the alternatively spliced type III connecting segment (IIICS). Using specific adhesive ligands and inhibitory probes, we have examined the role of each of these domains in fibronectin-mediated neurite extension of neurons from chick embryo dorsal root and sympathetic ganglia. In studies using explanted ganglia, both fl3, a 75-kD tryptic fragment of human plasma fibronectin containing the central cell-binding domain, and CS1-IgG, a synthetic peptide-IgG conjugate containing the principal cell adhesion site from the IIICS, supported neurite outgrowth after adsorption onto the substrate. The maximal activities of fl3 and CSl-IgG were 45-55% and 25-30% that of intact fibronectin, respectively. Co-coating of the substrate with f13 and CS1-IgG produced an additive stimulation of neurite outgrowth, the extent of which approached that obtained with fibronectin. Similar results were obtained with purified neuronal cell preparations isolated by tryptic dissociation of dorsal root ganglia. In complementary studies, blockage of the adhesive function of either the central cell-binding domain (with mAb 333, an antiadhesive monoclonal antibody) or the IIICS (with CS1 peptide), resulted in approximately 60 or 30% reduction in fibronectin-mediated neurite outgrowth, respectively. When tested in combination, the inhibitory activities of mAb 333 and CSl were additive. From these results, we conclude that neurons from the peripheral nervous system can extend neurites on both the central cell-binding domain and the IIICS region of fibronectin, and that these cells are therefore the first normal, embryonic cell type shown to adhere to the IIICS. These results suggest that spatiotemporal fluctuations in the alternative mRNA splicing of the IIICS region of fibronectin may be important in regulation of cell adhesive events during development of the peripheral nervous system.

Monoclonal antibody characterization of two distant sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration, and matrix assembly.

Site-directed mutagenesis studies have suggested that additional peptide information in the central cell-binding domain of fibronectin besides the minimal Arg-Gly-Asp (RGD) sequence is required for its full adhesive activity. The nature of this second, synergistic site was analyzed further by protein chemical and immunological approaches using biological assays for adhesion, migration, and matrix assembly. Fragments derived from the cell-binding domain were coupled covalently to plates, and their specific molar activities in mediating BHK cell spreading were compared with that of intact fibronectin. A 37-kD fragment purified from chymotryptic digests of human plasma fibronectin had essentially the same specific molar activity as intact fibronectin. In contrast, other fragments such as an 11.5-kD fragment lacking NH2-terminal sequences of the 37-kD fragment had only poor spreading activity on a molar basis. Furthermore, in competitive inhibition assays of fibronectin-mediated cell spreading, the 37-kD fragment was approximately 325-fold more active than the GRGDS synthetic peptide on a molar basis. mAbs were produced using the 37-kD protein as an immunogen and their epitopes were characterized. Two separate mAbs, one binding close to the RGD site and the other to a site approximately 15 kD distant from the RGD site, individually inhibited BHK cell spreading on fibronectin by greater than 90%. In contrast, an antibody that bound between these two sites had minimal inhibitory activity. The antibodies found to be inhibitory in cell spreading assays for BHK cells also inhibited both fibronectin-mediated cell spreading and migration of human HT-1080 cells, functions which were also dependent on function of the alpha 5 beta 1 integrin (fibronectin receptor). Assembly of endogenously synthesized fibronectin into an extracellular matrix was not significantly inhibited by most of the anti-37-kD mAbs, but was strongly inhibited only by the antibodies binding close to the RGD site or the putative synergy site. These results indicate that a second site distant from the RGD site on fibronectin is crucial for its full biological activity in diverse functions dependent on the alpha 5 beta 1 fibronectin receptor. This site is mapped by mAbs closer to the RGD site than previously expected.

Integrin function: molecular hierarchies of cytoskeletal and signaling molecules.

Integrin receptors play important roles in organizing the actin-containing cytoskeleton and in signal transduction from the extracellular matrix. The initial steps in integrin function can be analyzed experimentally using beads coated with ligands or anti-integrin antibodies to trigger rapid focal transmembrane responses. A hierarchy of transmembrane actions was identified in this study. Simple integrin aggregation triggered localized transmembrane accumulation of 20 signal transduction molecules, including RhoA, Rac1, Ras, Raf, MEK, ERK, and JNK. In contrast, out of eight cytoskeletal molecules tested, only tensin coaccumulated. Integrin aggregation alone was also sufficient to induce rapid activation of the JNK pathway, with kinetics of activation different from those of ERK. The tyrosine kinase inhibitors herbimycin A or genistein blocked both the accumulation of 19 out of 20 signal transduction molecules and JNK- and ERK-mediated signaling. Cytochalasin D had identical effects, whereas three other tyrosine kinase inhibitors did not. The sole exception among signaling molecules was the kinase pp125FAK which continued to coaggregate with alpha 5 beta 1 integrins even in the presence of these inhibitors. Tyrosine kinase inhibition also failed to block the ability of ligand occupancy plus integrin aggregation to trigger transmembrane accumulation of the three cytoskeletal molecules talin, alpha-actinin, and vinculin; these molecules accumulated even in the presence of cytochalasin D. However, it was necessary to fulfill all four conditions, i.e., integrin aggregation, integrin occupancy, tyrosine kinase activity, and actin cytoskeletal integrity, to achieve integrin-mediated focal accumulation of other cytoskeletal molecules including F-actin and paxillin. Integrins therefore mediate a transmembrane hierarchy of molecular responses.

Vinexin: a novel vinculin-binding protein with multiple SH3 domains enhances actin cytoskeletal organization.

Using the yeast two-hybrid system and an in vitro binding assay, we have identified a novel protein termed vinexin as a vinculin-binding protein. By Northern blotting, we identified two types of vinexin mRNA that were 3 and 2 kb in length. Screening for full-length cDNA clones and sequencing indicated that the two mRNA encode 82- and 37-kD polypeptides termed vinexin alpha and beta, respectively. Both forms of vinexin share a common carboxyl-terminal sequence containing three SH3 domains. The larger vinexin alpha contains an additional amino-terminal sequence. The interaction between vinexin and vinculin was mediated by two SH3 domains of vinexin and the proline-rich region of vinculin. When expressed, vinexin alpha and beta localized to focal adhesions in NIH 3T3 fibroblasts, and to cell-cell junctions in epithelial LLC-PK1 cells. Furthermore, expression of vinexin increased focal adhesion size. Vinexin alpha also promoted upregulation of actin stress fiber formation. In addition, cell lines stably expressing vinexin beta showed enhanced cell spreading on fibronectin. These data identify vinexin as a novel focal adhesion and cell- cell adhesion protein that binds via SH3 domains to the hinge region of vinculin, which can enhance actin cytoskeletal organization and cell spreading.

Novel function for beta 1 integrins in keratinocyte cell-cell interactions.

We have examined the expression, localization, and function of beta 1 integrins on cultured human epidermal keratinocytes using polyclonal and monoclonal antibodies against the beta 1, alpha 2, alpha 3, and alpha 5 integrin subunits. The beta 1 polypeptide, common to all class 1 integrins, was localized primarily in areas of cell-cell contacts of cultured keratinocytes, as were alpha 2 and alpha 3 polypeptides, suggesting a possible role in cell-cell adhesion for these integrin polypeptides. In contrast, the fibronectin receptor alpha 5 subunit showed no such accumulations in regions of cell-cell contact but was more diffusely distributed in the keratinocyte plasma membrane, consistent with the absence of fibronectin at cell-cell contact sites. Colonies of cultured keratinocytes could be dissociated by treatment with monoclonal antibody specific to the beta 1 polypeptide. Such dissociation of cell-cell contacts also occurred under conditions where the monoclonal antibody had no effect on cell-substrate adhesion. Therefore, beta 1 integrin-dependent cell-cell adhesion can be inhibited without affecting other cell-adhesive interactions. Antibody treatment of keratinocytes maintained in either low (0.15 mM) or high (1.2 mM) CaCl2 also resulted in the loss of organization of intracellular F-actin filaments and beta 1 integrins, even when the anti-beta 1 monoclonal antibody had no dissociating effect on keratinocyte colonies at the higher calcium concentration. Our results indicate that beta 1 integrins play roles in the maintenance of cell-cell contacts between keratinocytes and in the organization of intracellular microfilaments. They suggest that in epithelial cells integrins can function in cell-cell interactions as well as in cell-substrate adhesion.

Synergistic roles for receptor occupancy and aggregation in integrin transmembrane function.

Integrin receptors mediate cell adhesion, signal transduction, and cytoskeletal organization. How a single transmembrane receptor can fulfill multiple functions was clarified by comparing roles of receptor occupancy and aggregation. Integrin occupancy by monovalent ligand induced receptor redistribution, but minimal tyrosine phosphorylation signaling or cytoskeletal protein redistribution. Aggregation of integrins by noninhibitory monoclonal antibodies on beads induced intracellular accumulations of pp125FAK and tensin, as well as phosphorylation, but no accumulation of other cytoskeletal proteins such as talin. Combining antibody-mediated clustering with monovalent ligand occupancy induced accumulation of seven cytoskeletal proteins, including alpha-actinin, talin, and F-actin, thereby mimicking multivalent interactions with fibronectin or polyvalent peptides. Integrins therefore mediate a complex repertoire of functions through the distinct effects of receptor aggregation, receptor occupancy, or both together.

Specific increase in non-functional masseter bursts in subjects aware of tooth-clenching during wakefulness.

Previous studies have reported that subjective awareness of a tooth-clenching habit is associated with increased jaw motor activity (Rao SM, Glaros AG, J Dent Res. 1979;58:1872). The aim of this study was to test the hypothesis that subjects with clenching awareness exhibit different motor expressions specific to non-functional oromotor activity under laboratory conditions without psychological or sensory effects. Polygraphic and audio-video recordings were made for a 30-min period of silent reading by 33 subjects without oro-facial pain. Oro-facial behaviours (e.g. swallowing, lip movements) were scored according to the polygraphic and audio-video records and masseter bursts were quantitatively analysed. Subjective psychological/sensory measures were also recorded before and/or after the polygraphic recording using a visual analogue scale. The subjects were classified into two groups one with 15 subjects who were aware of having a tooth-clenching habit and another with 18 who were not aware of any such habit. There were no differences between the groups with respect to the number of functional oro-facial behaviours or subjective psychological/sensory measures. Masseter bursts unrelated to functional oro-facial behaviours occurred more frequently in subjects with awareness [median (range) = 23 (2-187) bursts] than in those without [9.0 (0-36); P = 0.01], while neither burst activity [12.3 (1.8-34.5) % of maximum voluntary clenching and 10.1 (6.5-25.1) %, respectively] nor duration [1.17 (0.2-2.2) s and 1.28 (0.3-4.1) s, respectively] differed between the groups. The occurrence of functional oro-facial behaviours or other body behaviours (e.g. limb and body movements) did not differ between the two groups. These findings suggest that the increased masseter activity in subjects with tooth-clenching awareness is characterized by a specific increase in non-functional masseter bursts.