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High levels of circulating lipopolysaccharide (LPS) cause intestinal inflammation and increased permeability to bacteria and toxins. To better understand the effects of LPS on the gut, confocal microscopy and immunofluorescence staining were used to examine the distribution of LPS in the rat intestine after intravenous or enteral administration. LPS was localized in macrophages in the lamina propria from 1 h to >28 days after intravenous injection. LPS was also detected in the epithelial cells from 8 h to 7 days after injection. In contrast, LPS administered enterally was found in the gut lumen in close proximity to the mucosa but was not detected in enterocytes at any time. The concentration of LPS in enterocytes near the villus tip provides a mechanism for the clearance of endotoxin, by the turnover and shedding of LPS-containing enterocytes into the gut lumen, that has not been previously described.

Using amino acids 884-1066 and 884-1012 expressed from chicken vinculin as fusion proteins with schistosomal glutathione S-transferase, we determined the binding kinetics of the protein fragments with F-actin. We established by the stopped-flow method a two-step binding process: an initial rapid reaction followed by a slower process. The latter is attributed to F-actin cross-linking and/or bundling, which was previously detected by viscometry and electron microscopy [Johnson, R. P. & Craig, S. W. (1995) Nature 373, 261-264]. This is also supported by dynamic light-scattering measurements, indicating dramatic changes in the internal actin filament dynamics, i.e. in bending undulations due to thermal noise. The similar size of the binding reaction for both fusion proteins with F-actin indicates that the F-actin binding site(s) on vinculin are located between residues 884-1012. No binding of pure glutathione S-transferase or its fusion protein with vinculin peptide 1012-1066 with F-actin was detected by either method.

We studied the elasticity of both a wild type (F9) mouse embryonic carcinoma and a vinculin-deficient (5.51) cell line, which was produced by chemical mutagenesis. Using cell poking, we measured the effects of loss of vinculin on the elastic properties of these cells. F9 cells were about 20% more resistant to indentation by the cell poker (a glass stylus) than were 5.51 cells. Using the atomic force microscope to map the elasticity of wild type and vinculin-deficient cells by 128 X 128 force scans, we observed a correlation of elasticity with cell poking elastometric measurements. These findings, as well as previous atomic force, rheologic, and magnetometric measurements [Goldmann and Ezzell, Exp. Cell Res. 226 (1996) 234-237; Ezzell et al., Exp. Cell Res. 231 (1997) 14-26], indicate that vinculin is an integral part of the cytoskeletal network.

We examined the binding kinetics of intact talin and talin head and tail fragment with F-actin at pH 7.0 and at low ionic strength. We observed by a transient kinetic method a fast followed by a slower binding process for intact talin and talin tail fragment with filamentous actin. The latter can be attributed to F-actin cross-linking and/or bundling, which was observed in cosedimentation assays as well as by low shear viscometry and electron microscopy [Zhang, J., Robson, R. M., Schmidt, J. M. & Stromer, M. H. (1996) Biochem. Biophys. Res. Commun. 218, 530-537]. This finding is supported by dynamic light scattering measurements, indicating changes in internal actin filament dynamics due to cross-linking/bundling events with intact talin and talin tail fragment. No binding of the talin head fragment with F-actin was detected by either method.

Distributions of immunoreactive interleukin-1 (IL-1) and lipopolysaccharide (LPS) were studied in the tissues of rats after intravenous injection of purified LPS or live Escherichia coli bacteria. IL-1 staining in the spleen peaked at 4-8 h, colocalized with LPS in marginal zone macrophages, and was undetectable 24 h after injection, whereas LPS staining peaked at 24 h and was detectable for 4 weeks. The tissue IL-1 response was similar for LPS and live bacteria. Thus, tissue IL-1 is down-regulated within hours despite maintenance of LPS in the same cells for weeks. Macrophages in liver and lung had only slight IL-1 staining despite intense staining for LPS. Tissue IL-1 production appears to be differentially regulated after gram-negative bacteremia; LPS cleared by liver and lung macrophages elicit minimal IL-1, whereas there is high local IL-1 production in the marginal zone of the spleen that may increase immune responses to bacterial wall antigens.

Cross-linking of actin filaments by filamin by means of frequency-dependent rheology yields an increase in the filament's elasticity and stiffness. Higher cross-linker (filamin) ratios are required for mean actin-filament lengths of 5-6 microm than for random-length distribution of actin filaments. The loss modulus (i.e. the viscous portion) in the region of the internal-chain dynamics [G"(omega) approximately omega(alpha)] is influenced by the cross-linking of filaments, and with an increasing molar ratio of filamin/actin a reduction of alpha is observed. Rheological measurements reveal that actin networks are already formed at the polymerizing stage at a molar ratio of filamin/actin of less than 1:100, and electron micrographs show phase separation of actin/filament networks of low density and of actin/filament bundles.

The author introduces some of the basic concepts in the theory and mechanics of digital signal processing techniques as applied to microscopy. With the right software and a sound approach, a great deal of information can be retrieved from noisy or degraded images. The author also discusses manipulation of images in order to produce a clearer images for the observer.

Müllerian inhibiting substance (MIS) is a glycoprotein hormone required for normal male reproductive tract development; it is presumed to signal through a heteromeric complex of type I and type II receptors. MIS exposure produces a paracrine-mediated regression of the embryonic Müllerian duct with histological changes consistent with apoptosis. MIS has also been shown to inhibit fetal lung development in vitro and in vivo, although the mechanism of this inhibition is unknown. The primordial lung and gonad are anatomically proximate on embryonic day 13.5, raising the possibility of a paracrine-mediated influence of MIS in male embryos on lung as well as MIS effecting dissolution of the Müllerian duct. We hypothesized that a negative regulatory event(s) might occur in the lung, as occurs in the duct, at the onset of MIS protein expression; thus, apoptosis and branching morphogenesis were studied in explanted fetal rat lungs incubated with proteolytically activated MIS. MIS exposure resulted in reduced total lung bud number as well as lung perimeter length. Explanted lungs exposed to MIS also exhibited numerous apoptotic bodies. To assess whether this MIS-induced phenomenon in lung might be mediated by the MIS type II receptor (MIS RII), reverse transcriptase-PCR performed on multiple fetal rat lung RNA samples using oligonucleotide primers designed from the 3'-untranslated region of rat MIS RII complementary DNA showed a product of the expected size that when sequenced was nearly identical to rat MIS RII. Northern blot analysis using polyadenylated fetal rat lung RNA and a 3'-MIS RII probe revealed a 2-kilobase transcript that was also seen in testicular messenger RNA. These studies show that the putative ligand binding receptor for MIS is expressed in embryonic lung, where MIS negatively modulates branching and activates apoptosis. We speculate that the mechanism of MIS-induced inhibition of lung development in the male fetus begins with MIS binding to the MIS RII, followed by a signaling cascade resulting in delayed airway branching temporally associated with enhanced apoptosis.

Disruptions in the mucosal lining of the gastrointestinal tract reseal by epithelial cell migration, a process termed restitution. We examined the involvement of laminin isoforms and their integrin receptors in restitution using the intestinal epithelial cell line T84. T84 cells express primarily laminins 5, 6, and 7 as indicated by immunostaining using laminin subunit-specific monoclonal antibodies (MAbs). A MAb (BM2) specific for the laminin alpha 3 subunit, a component of laminins 5, 6, and 7, completely inhibited the closure of mechanical wounds in T84 monolayers. Confocal microscopy using MAbs BM2 (laminin alpha 3 subunit) and 6F12 (laminin beta 3 subunit) revealed that laminin-5 is deposited in a basal matrix that extends into the wound. The MAbs 4E10 (laminin beta 1 subunit) and C4 (laminin beta 2 subunit) stained the lateral membranes between T84 cells. This staining was enhanced in cells adjoining wounds. Because T84 cells stained faintly with MAbs 4C7 (laminin alpha 1 subunit) and with MAbs 4F11 and 1B4 (laminin alpha 2 subunit), we suggest that expression of laminins 6 and 7 is enhanced in response to wounding. The alpha 3 beta 1 integrin and the alpha 6-containing integrins function in wound closure because MAbs specific for the beta 1 integrin subunit (MAb13), the alpha 3 subunit (IVA5), and the alpha 6 subunit (2B7) potently inhibited T84 migration into wounds. Immunofluorescence using UMA9, a beta 4-integrin-specific MAb, revealed that alpha 6 beta 4 integrin exists in a Triton-X-100-insoluble structure at the basal surface and that the staining of this structure is enhanced in cells adjoining wounds. In addition, a Triton-X-100-soluble pool of alpha 6 beta 4, as well as alpha 3 beta 1 and presumably alpha 6 beta 1, was found along lateral surfaces of T84 cells. On flattened cells adjoining wounds, staining for these integrins was distributed diffusely, suggesting a redistribution that accompanies cell migration. Taken together, these data suggest that wound-induced epithelial cell migration is a finely tuned process that is dependent upon the regulated function and localization of specific laminins and their integrin receptors.

We have been studying mouse F9 embryonic carcinoma cells which contain no detectable vinculin protein (5.51 cells), and compared them with F9 wild-type cells. Employing atomic force microscopy, we probed the elastic properties of individual F9 wild-type and 5.51 cells by measuring the dynamic response of controlled loads of the cantilever tip. An elastic modulus (Young) of approximately 3.8 and approximately 2.5 kPa was calculated for wild-type and 5.51 cells, respectively. Using disc rheometry, we detected a marked change in shear of a 1000g pellet of approximately 55 x 10(6) cells between wild-type and 5.51 mutants. These differences are attributed to the loss of vinculin and altered cytoskeletal organization in these cells.

We have studied the motility of wild-type F9 and vinculin-deficient (5.51) mouse embryonal carcinoma cells. F9 cells extended filopodia at a rate of 61 ( +/- 18) nm/s over a distance of 3.18 (+/- 0.29) microns. In contrast, 5.51 cells exhibited filopodia which extended at a similar speed of 57 (+/- 17) nm/s but over a longer distance of 5.10 (+/- 2.14) microns. Cell-substratum contact areas of both cell types were examined by reflection interference contrast microscopy. Wild-type F9 cells had distinct close contacts (dark gray areas) at the cell periphery, whereas 5.51 cells had only a few light gray pinpoint contacts with the substrate. Confocal microscopy showed alpha-actinin to be localized along actin stress fibers in wild-type cells, and in 5.51 cells stress fibers were absent and alpha-actinin was associated with F-actin in the filopodia. beta 1-integrin, talin, and paxillin were concentrated in focal contacts in wild-type cells, but in 5.51 cells beta 1-integrin and talin were in patches under the plasma membrane and paxillin was diffusely distributed in the cytoplasm. We conclude that changes in cell shape and motility of 5.51 compared to wild-type F9 cells are due to the absence of vinculin even though there may be functions of other focal adhesion complex proteins, e.g., talin, linking the actin cytoskeleton to the plasma membrane.