An IKLLI-containing peptide derived from the laminin alpha1 chain mediating heparin-binding, cell adhesion, neurite outgrowth and proliferation, represents a binding site for integrin alpha3beta1 and heparan sulphate proteoglycan.
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We synthesized and characterized several peptides containing the IKLLI sequence in the alpha1 chain of laminin-1. The IKLLI-containing peptides, such as LA4 (CSRNLSEIKLLISRARK), LA5 (EIKLLIS) and LA5L (SEIKLLIS), were found to mediate heparin binding and cell adhesion, while also promoting neurite outgrowth in PC12 cells. Furthermore, peptides LA4 and LA5 also mediated proliferation. However, a scrambled peptide, LA5S (ILEKSLI), did not show any of these activities. Anti-LA4 antibodies inhibited laminin- and LA5-mediated cell adhesion and neurite outgrowth, and anti-(integrin alpha3) and anti-(integrin beta1) antibodies inhibited LA5-mediated cell adhesion and neurite outgrowth. Heparin and heparan sulphate inhibited LA5-mediated heparin binding and PC12 cell adhesion in a dose- dependent manner. The IC50 for inhibition of heparin binding and cell adhesion was observed with 9 microM and 8 microM heparin/heparan sulphate respectively. Furthermore, heparan sulphate proteoglycan also inhibited LA5-mediated PC12 cell adhesion with an IC50 of 100 micrograms/ml. However, chondroitin sulphate (dermatan sulphate) did not inhibit cell adhesion. These data suggest that an IKLLI-containing peptide derived from the laminin alpha1 chain may be an active site of laminin and that its cell adhesion may thus interact with both integrin alpha3beta1 and cell- surface heparan sulphate proteoglycan. (+info)
Growth plate cartilage formation and resorption are differentially depressed in growth retarded uremic rats.
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To characterize the modifications of growth plate in individuals with growth impairment secondary to chronic renal failure, young rats were made uremic by subtotal nephrectomy (NX) and, after 14 d, their tibial growth plates were studied and compared with those of sham-operated rats fed ad libitum (SAL) or pair-fed with NX (SPF). NX rats were growth retarded and severely uremic. Growth plate height (mean +/- SD) was much greater (P<0.05) in NX (868.4+/-85.4 microm) than SAL (570.1+/-93.5 microm) and SPF (551.9+/-99.7 microm) rats as a result of a higher (P<0.05) hypertrophic zone (661.0+/-89.7 versus 362.8+/-71.6 and 353.0+/-93.9 microm, respectively). The increased size of the growth plate was associated with a greater number of chondrocytes and modifications in their structure, particularly in the hypertrophic zone adjacent to bone. In this zone, chondrocytes of NX animals were significantly (P<0.05) smaller (12080.4+/-1158.3 microm3) and shorter (34.1+/-2.5 microm) than those of SAL (16302.8+/-1483.4 microm3 and 37.8+/-2.0 microm) and SPF (14465.8+/-1521.0 microm3 and 36.3+/-1.8 microm). The interface between the growth plate cartilage and the metaphyseal bone appeared markedly irregular in NX rats. Kinetics of chondrocytes was also modified (P<0.05) in the NX rats, which had lower cell turnover per column per day (5.4+/-0.9), longer duration of hypertrophic phase (89.0+/-15.2 h), and reduced cellular advance velocity (7.4+/-2.2 microm/h) compared with SAL (8.0+/-1.6, 32.1+/-6.7 h, and 11.3+/-2.7 microm/h) and SPF (7.2+/-1.1, 34.8+/-5.1 h, and 10.1+/-2.5 microm/h). Cell proliferation was no different among the three groups. Because the growth plates of SPF and SAL rats were substantially not different, modifications observed in the NX rats cannot be attributed to the nutritional deficit associated with renal failure. These findings indicate that chronic renal failure depresses both the activity of the growth plate cartilage by altering chondrocyte hypertrophy and the replacement of cartilage by bone at the metaphyseal end. The two processes are differentially depressed since cartilage resorption is more severely lowered than cartilage enlargement and this leads to an accumulation of cartilage at the hypertrophic zone. (+info)
Effect of ultrasonically nebulized distilled water on airway epithelial cell swelling in guinea pigs.
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To investigate the pathogenesis of ultrasonically nebulized distilled water-induced airway narrowing, we studied the role of airway epithelial cells during a distilled water-inhalation challenge in an animal model of airway inflammation. Guinea pigs were divided into four groups: 1) a sham/saline (S/S) group: sham ozone followed by saline inhalation; 2) a sham/water (S/W) group: sham ozone followed by water inhalation; 3) an ozone/saline (O/S) group: ozone followed by saline inhalation; and 4) an ozone/water (O/W) group: ozone followed by water inhalation. After exposure to either 3.0 parts/million ozone or air at the same flow rate for 2 h, guinea pigs were anesthetized and tracheostomized, and then lung resistance (RL) was measured. For morphometric assessment, tissues were fixed with formaldehyde, stained with hematoxylin and eosin, and cut into transverse sections. Airway dimensions were either measured directly or calculated from the internal perimeter, the external perimeter, and airway wall area. There were no statistical differences in the values of RL before distilled water inhalation between the sham groups and the ozone groups. RL increased significantly after 10 min of distilled water inhalation in both the S/W group and the O/W group. In the S/W group, epithelial cells were swollen, and intercellular spaces were wider, resulting in significant increase in epithelial wall thickness, but there was no significant infiltration by inflammatory cells. In the O/S group, the epithelium showed infiltration by inflammatory cells without change in cell volume. In the O/W group, the epithelium showed both infiltration and a greater increase in epithelial wall thickness compared with the S/W group. These results suggest that airway epithelial cell swelling, induced by inhaled distilled water, increases with RL in guinea pigs and that this reaction may be accelerated by airway inflammation. (+info)
Osmotically induced cell volume changes alter anterograde and retrograde transport, Golgi structure, and COPI dissociation.
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Physiological conditions that impinge on constitutive traffic and affect organelle structure are not known. We report that osmotically induced cell volume changes, which are known to occur under a variety of conditions, rapidly inhibited endoplasmic reticulum (ER)-to-Golgi transport in mammalian cells. Both ER export and ER Golgi intermediate compartment (ERGIC)-to-Golgi trafficking steps were blocked, but retrograde transport was active, and it mediated ERGIC and Golgi collapse into the ER. Extensive tubulation and relatively rapid Golgi resident redistribution were observed under hypo-osmotic conditions, whereas a slower redistribution of the same markers, without apparent tubulation, was observed under hyperosmotic conditions. The osmotic stress response correlated with the perturbation of COPI function, because both hypo- and hyperosmotic conditions slowed brefeldin A-induced dissociation of betaCOP from Golgi membranes. Remarkably, Golgi residents reemerged after several hours of sustained incubation in hypotonic or hypertonic medium. Reemergence was independent of new protein synthesis but required PKC, an activity known to mediate cell volume recovery. Taken together these results indicate the existence of a coupling between cell volume and constitutive traffic that impacts organelle structure through independent effects on anterograde and retrograde flow and that involves, in part, modulation of COPI function. (+info)
Primary mesenchymal cells isolated from SPARC-null mice exhibit altered morphology and rates of proliferation.
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SPARC (secreted protein acidic and rich in cysteine)/BM 40/osteonectin is a matricellular protein shown to function as a counteradhesive factor that induces cell rounding and as an inhibitor of cell proliferation. These activities have been defined in cell culture, in which interpretation has been complicated by the presence of endogenous SPARC. We therefore sought to determine whether cell shape and proliferation would be affected by the absence of SPARC. Mesangial cells, fibroblasts, and aortic smooth muscle cells were isolated from SPARC-null and age-matched, wild-type mice. In contrast to wild-type cells, SPARC-null mesangial cells exhibited a flat morphology and an altered actin cytoskeleton. In addition, vinculin-containing focal adhesions were distributed over the center of SPARC-null cells, whereas in wild-type cells, the number of focal adhesions was reduced, and these structures were restricted largely to the cell periphery. Although the SPARC-null fibroblasts did not display overt differences in cell morphology, the cells responded to exogenous recombinant SPARC by rounding up in a manner similar to that of wild-type fibroblasts. Thus, the expression of endogenous SPARC is not required for the response of cells to SPARC. Additionally, SPARC-null mesangial cells, fibroblasts, and smooth muscle cells proliferated faster than their respective wild-type counterparts. Null cells also showed a greater sensitivity to the inhibition of cell cycle progression by the addition of recombinant SPARC. The increased proliferation rate of SPARC-null cells appeared to be mediated, at least in part, by an increase in the cell cycle regulatory protein cyclin A. We conclude that the expression of SPARC influences the cellular architecture of mesangial cells and that SPARC plays a role in the regulation of cell cycle in mesangial cells, fibroblasts, and smooth muscle cells. (+info)
A betaPP peptide carboxyl-terminal to Abeta is neurotoxic.
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Extracellular Abeta-amyloid and intraneuronal paired helical filaments (PHFs) composed of tau protein are the neuropathological hallmark of Alzheimer's disease. Abeta is a 39- to 43-residue peptide derived by cleavage of a 695- to 770-amino-acid membrane-associate glycoprotein (termed beta-protein precursor, betaPP). Following the observation that an antiserum to an epitope located between residues 713 and 723 of betaPP770 (ie, the transmembrane region of the betaPP distal to Abeta) labels PHFs and that a synthetic peptide homologous to residues 713 to 730 of betaPP770 (betaPP713-730) is highly fibrillogenic and interacts with tau in vitro, it has been hypothesized that betaPP fragments other than Abeta may feature in the pathogenesis of Alzheimer's disease concurring with neuronal degeneration. To investigate this issue, we have analyzed the effects of the exposure of primary neuronal cultures to the synthetic peptide betaPP713-730. Cultures were prepared from rat hippocampus on embryonic day 17 and incubated with the peptide at 2.5 to 30 micromol/L concentration for 1 to 4 days. Cell viability was compared with that of control cultures exposed to a scrambled sequence of the peptide. A 4-day exposure to 20 micromol/L betaPP713-730 resulted in almost complete neuronal loss, whereas no changes were observed with the scrambled peptide. Degenerating neurons showed DNA fragmentation by agarose gel electrophoresis and apoptotic changes by light and electron microscopy. These findings support the view that betaPP sequences other than Abeta may play a role in nerve cell degeneration in Alzheimer's disease. (+info)
Expression of matrix metalloprotease-2-cleaved laminin-5 in breast remodeling stimulated by sex steroids.
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The extracellular matrix plays an important role in breast remodeling. We have shown that matrix metalloprotease-2 (MMP2) cleaves laminin-5 (Ln-5), a basement membrane component, generating a fragment called gamma2x. Human breast epithelial cells, while constitutively immobile on intact Ln-5, acquire a motile phenotype on MMP2-cleaved Ln-5. We hypothesize that this mechanism may underlie cell mobilization across the basement membrane during branching morphogenesis in breast development regulated by sex steroids. We report that the expression of MMP2 and cleavage of Ln-5 correlate well with tissue remodeling and epithelial rearrangement of the breast both in vivo and in vitro. Thus, the Ln-5 gamma2x fragment was detected by immunoblotting in sexually mature, pregnant, and postweaning, but not in prepubertal or lactating mammary glands. Furthermore, cleaved Ln-5, as well as MMP2, became detectable in remodeling glands from sexually immature rats treated with sex steroids. In rat mammary gland explants, epithelial reorganization and luminal cell morphological changes were induced by the addition of exogenous MMP2, in parallel to the appearance of cleaved Ln-5. Similar effects were observed in epithelial monolayers plated on human Ln-5 and exposed to MMP2. These results suggest that cleavage of Ln-5 by MMP2 might be regulated by sex steroids and that it may contribute to breast remodeling under physiological and possibly pathological conditions. (+info)
Transient and sustained increases in inositol 1,4,5-trisphosphate precede the differential growth response in gravistimulated maize pulvini.
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The internodal maize pulvinus responds to gravistimulation with differential cell elongation on the lower side. As the site of both graviperception and response, the pulvinus is an ideal system to study how organisms sense changes in orientation. We observed a transient 5-fold increase in inositol 1,4,5-trisphosphate (IP3) within 10 s of gravistimulation in the lower half of the pulvinus, indicating that the positional change was sensed immediately. Over the first 30 min, rapid IP3 fluctuations were observed between the upper and lower halves. Maize plants require a presentation time of between 2 and 4 h before the cells on the lower side of the pulvinus are committed to elongation. After 2 h of gravistimulation, the lower half consistently had higher IP3, and IP3 levels on the lower side continued to increase up to approximately 5-fold over basal levels before visible growth. As bending became visible after 8-10 h, IP3 levels returned to basal values. Additionally, phosphatidylinositol 4-phosphate 5-kinase activity in the lower pulvinus half increased transiently within 10 min of gravistimulation, suggesting that the increased IP3 production was accompanied by an up-regulation of phosphatidylinositol 4, 5-bisphosphate biosynthesis. Neither IP3 levels nor phosphatidylinositol 4-phosphate 5-kinase activity changed in pulvini halves from vertical control plants. Our data indicate the involvement of IP3 and inositol phospholipids in both short- and long-term responses to gravistimulation. As a diffusible second messenger, IP3 provides a mechanism to transmit and amplify the signal from the perceiving to the responding cells in the pulvinus, coordinating a synchronized growth response. (+info)