Effect of actomyosin contractility on focal contacts of myofibroblasts and structure of stress fibers

Myofibroblasts from rat lung were cultivated. These cells in addition to beta- and gamma-cytoplasmic actins, expressed alpha-smooth muscle actin (alpha-SMA) and formed a system of "supermature" focal contacts, which were connected with thick stress-fibers expressing alpha-SMA and myosin II. Reduction of actin-myison contractility by inhibitors BDM and ML-7 lead to stress fiber reorganization, e.g., decrease in their thickness, a selective disappearance of alpha-SMA expression and myosin translocation from bundles to the cytoplasm. Using immunofluorescence, interference-reflection microscopy and morphometry, we have demonstrated that an inhibition of actin-myosin contractility also leads to dispersion of myofibroblastic focal contacts. Phase-contrast and DIC video-enhanced microscopy of live cells showed morphological reorganization at the leading edge after inhibitory treatment. Thus, actin-myosin contractility controls the structure of "supermature" focal contacts of myofibroblasts and alpha-SMA expression in stress fibers.     

Synthetic peptide GRGDS induces dissociation of alpha-actinin and vinculin from the sites of focal contacts

The synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS) mimics the cellular binding site of many adhesive proteins in the extracellular matrix and causes rounding and detachment of spread cells. We have studied whether its binding affects the associations of two major components, alpha-actinin and vinculin, at the adhesion plaque. Living 3T3 cells were microinjected with fluorescently labeled alpha-actinin and/or vinculin and observed using video microscopy before and after the addition of 50 micrograms/ml GRGDS. As soon as 5 min after treatment, fluorescent alpha-actinin and vinculin became dissociated simultaneously from the sites of many focal contacts. The proteins either moved away as discrete structures or dispersed from adhesion plaques. As a result, the enrichment of alpha-actinin and vinculin at these focal contacts was no longer detected. The focal contacts then faded away slowly without showing detectable movement. These data suggest that the binding state of integrin has a transmembrane effect on the distribution of cytoskeletal components. The dissociation of alpha-actinin and vinculin from adhesion plaques may in turn weaken the contacts and result in rounding and detachment of cells.     

Vasoactive intestinal peptide in the normal and inflamed feline gallbladder

Intravenous infusion of vasoactive intestinal peptide (VIP) causes gallbladder mucosal fluid secretion by an action on epithelial cell receptors in the cat. Gallbladder fluid secretion is observed also in experimental cholecystitis and this secretion is abolished when the intramural gallbladder nerves are blocked. In the present study, immunoreactive VIP was detected in the gallbladder contents (29 +/- 5 (S.E.M.) pM) in the obstructed lumen of the gallbladder in cats with experimental cholecystitis and gallbladder mucosal fluid secretion, but not in the normal feline gallbladder. During luminal perfusion of the gallbladder in vivo, the calculated secretion of VIP into the gallbladder lumen in animals with experimental cholecystitis was significantly higher (0.31 +/- 0.08 (S.E.M.), pmol/h) than in controls (0.11 +/- 0.02 (S.E.M.), pmol/h) while plasma levels of VIP were similar. Recovery of exogenously administered VIP was similar in normal and inflamed gallbladders. The present results support the hypothesis that intramural VIP-releasing nerve fibers may be activated in cholecystitis.     

Coliforms and enterococci isolated from the intestinal tract of conventional mice

Coliforms and enterococci were isolated from the intestinal tract of infant (12-day-old) and adult (6-to 8-week-old) conventional mice. Eighty coliform isolates and eighty enterococcal strains were grouped according to their ability to ferment or hydrolyze various substrates. Sixty-one of the coliform isolates were identified asEscherichia coli. The remaining 19 strains were similar toE. coli, but did not produce-galactosidase. The enterococci belonged to two species:Streptococcus faecium andS. faecalis. Four biotypes ofS. faecium and two biotypes ofS. faecalis were detected. Xylosefermenting enterococci were isolated with a higher frequency from infant mice than from adults.     

Focal contacts of normal and RSV-transformed quail cells : Hypothesis of the transformation-induced deficient maturation of focal contacts

Morphology and distribution of cell-substrate contacts and their association with microfilament bundles in normal and RSV-transformed quail fibroblasts (16Q line) were studied by indirect immunofluorescence. The focal contacts were visualized by antibody exclusion method using monoclonal antibody to 80 kD serum protein adsorbed on the substratum. Embryo quail cells formed focal contacts of two morphological types: (1) small punctate; and (2) elongated large contacts. These two variants of contacts were designated respectively as dot and dash contacts. Both of focal contacts contained vinculin and alpha-actinin. Double immunofluorescence staining with polyclonal antibody to actin and monoclonal antibody to vinculin revealed that the dot contacts, in contrast to the dash ones, were not associated with microfilament bundles. The dot contacts were localized mostly near the active cell edges, while the dash contacts were found near the retracted cell edges and also under the central parts of the cell. We suppose that dot contacts represent initial structures which then can undergo maturation transforming them into dash contacts. RSV-transformed 16Q cells had predominantly the dot contacts which were not only located at the edges but also in the more central parts of the lamella. The dash contacts were present only in the minority of 16Q cells. RSV transformation is assumed to affect not the ability of cells to form initial dot contacts, but the maturation of dot contacts into dash contacts.     

Differential expression of TGF-beta isoforms by normal and inflammatory bowel disease intestinal myofibroblasts

First publishedSeptember 5, 2001; 10.1152/ajpcell. 00048.2001.Intestinalstrictures are frequent in Crohn's disease but not ulcerative colitis.We investigated the expression of transforming growth factor (TGF)-isoforms by isolated and cultured primary human intestinalmyofibroblasts and the responsiveness of these cells and intestinalepithelial cells to TGF- isoforms. Normal intestinal myofibroblastsreleased predominantly TGF-₃ and ulcerative colitismyofibroblasts expressed both TGF-₁ andTGF-₃, whereas in myofibroblast cultures from fibroticCrohn's disease tissue, there was significantly lower expression ofTGF-₃ but enhanced release of TGF-₂.These distinctive patterns of TGF- isoform release were sustainedthrough several myofibroblast passages. Proliferation of Crohn'sdisease myofibroblasts was significantly greater than that ofmyofibroblasts derived from normal and ulcerative colitis tissue. Incontrast to cells from normal and ulcerative colitis tissue,neutralization of the three TGF- isoforms did not affect theproliferation of Crohn's disease intestinal myofibroblasts. Studies onthe effect of recombinant TGF- isoforms on epithelial restitutionand proliferation suggest that TGF-₂ may be the least effective of the three isoforms in intestinal wound repair. In conclusion, the enhanced release of TGF-₂ but reducedexpression of TGF-₃ by Crohn's disease intestinalmyofibroblasts, together with their enhanced proliferative capacity,may lead to the development of intestinal strictures.

     

Lactobacillus alvi sp. nov., isolated from the intestinal tract of chicken

Strain R54(T) was isolated from the gizzard of hens. The isolate was Gram-positive, facultative anaerobic, gas-forming, catalase-negative, nonmotile, nonspore-forming and short-rod-shaped. The optimal temperature for growth was 40 °C and the DNA G+C content was 42.7 mol%. The 16S rRNA gene sequences similarity showed that strain R54(T) was most closely related to Lactobacillus ingluviei LMG 20380(T) (97.5%), followed by Lactobacillus coleohominis CIP 106820(T) (96.1%), Lactobacillus secaliphilus DSM 17896(T) (95.6%) and Lactobacillus gastricus LMG 22113(T) (95.4%). The DNA-DNA relatedness between strain R54(T) and L. ingluviei LMG 20380(T) , was 43.3%. The predominant cellular fatty acids of strain R54(T) were C(18:1 ω9c) (64.9%) and C(16:0) (20.0%), and the major polar lipid group was phospholipids. On the basis of polyphasic taxonomy approach, strain R54(T) represents a novel species of the genus Lactobacillus, for which the name Lactobacillus alvi sp. nov. is proposed (type strain R54(T) = KCCM 90099(T) = JCM 17644(T)).     

The antimicrobial effects of glucosinolates and their respective enzymatic hydrolysis products on bacteria isolated from the human intestinal tract

Aims:  The aim of the study was to evaluate the in vitro antibacterial activity of glucosinolates and their enzymatic hydrolysis product against bacteria isolated from the human intestinal tract.
Methods and results:  Using a disc diffusion bioassay, different doses of intact glucosinolates and their corresponding hydrolysis products were tested. There were clear structure–activity and concentration differences with respect to the in vitro growth inhibition effects as well as differences in the sensitivities of the individual bacteria. The most effective glucosinolate hydrolysis products were the isothiocyanates; sulforaphane and benzyl isothiocyanate were the best inhibitors of growth. Indole-3-carbinol had some inhibitory effects against the Gram-positive bacteria but had no effect, even at the highest dose, against the Gram-negative bacteria. Indole-3-acetonitrile had some inhibitory activity against the Gram-negative bacteria. Glucosinolates, nitriles and amines were ineffective at all the doses used.
Conclusions:  Glucosinolate hydrolysis products and specifically the isothiocyanates SFN and BITC have significant antimicrobial activity against Gram-positive and Gram-negative bacteria, and might be useful in controlling human pathogens through the diet.
Significance and Impact of the Study:  This the first major in vitro study demonstrating the potential of these natural dietary chemicals as an alternative to, or in combination with, current antibiotic-based therapies for treating infectious diseases.     

Characteristics of a new cellulolytic Clostridium sp. isolated from pig intestinal tract.

Gram-positive, spore-forming, motile, cellulolytic rods were isolated from 10(7) dilutions of pig fecal samples. The pigs had previously been fed pure cultures of the ruminal cellulolytic organism Clostridium longisporum. Isolates formed terminal to subterminal spores, and a fermentable carbohydrate was required for growth. Besides cellulose, the isolates utilized cellobiose, glycogen, maltose, and starch. However, glucose, fructose, sucrose, pectin, and xylose were not used as energy sources. Major fermentation products included formate and butyrate. The isolates did not digest proteins from gelatin or milk. Unlike C. longisporum, which has limited ability to degrade cell wall components from grasses (switchgrass, bromegrass, and ryegrass), the swine isolates were equally effective in degrading these components from both alfalfa and grasses. The extent of degradation was equal to or better than that observed with the predominant ruminal cellulolytic organisms. On the basis of morphology, motility, spore formation, fermentation products, and the ability to hydrolyze cellulose, the isolates are considered to be a new species of the genus Clostridium. It is unclear whether C. longisporum played a role in the establishment or occurrence of this newly described cellulolytic species. This is the first report of a cellulolytic Clostridium sp. isolated from the pig intestinal tract.     

Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism

The transition of cell-matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II-driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein-tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. Nat. Cell Biol. 1:136-143), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force.     

Characteristics of a new cellulolytic Clostridium sp. isolated from pig intestinal tract.

Gram-positive, spore-forming, motile, cellulolytic rods were isolated from 10(7) dilutions of pig fecal samples. The pigs had previously been fed pure cultures of the ruminal cellulolytic organism Clostridium longisporum. Isolates formed terminal to subterminal spores, and a fermentable carbohydrate was required for growth. Besides cellulose, the isolates utilized cellobiose, glycogen, maltose, and starch. However, glucose, fructose, sucrose, pectin, and xylose were not used as energy sources. Major fermentation products included formate and butyrate. The isolates did not digest proteins from gelatin or milk. Unlike C. longisporum, which has limited ability to degrade cell wall components from grasses (switchgrass, bromegrass, and ryegrass), the swine isolates were equally effective in degrading these components from both alfalfa and grasses. The extent of degradation was equal to or better than that observed with the predominant ruminal cellulolytic organisms. On the basis of morphology, motility, spore formation, fermentation products, and the ability to hydrolyze cellulose, the isolates are considered to be a new species of the genus Clostridium. It is unclear whether C. longisporum played a role in the establishment or occurrence of this newly described cellulolytic species. This is the first report of a cellulolytic Clostridium sp. isolated from the pig intestinal tract.     

Myocilin promotes substrate adhesion,spreading and formation of focal contacts in podocytes and mesangial cells

Myocilin, a secreted glycoprotein of the olfactomedin family, is constitutively expressed in podocytes of the rat kidney and induced in mesangial cells during mesangioproliferative glomerulonephritis. As myocilin has been found to be associated with fibrillar components of the extracellular matrix, and adhesive properties have been shown for other members of the olfactomedin family, we hypothesized that myocilin might play a role in cell-matrix interactions in the glomerulus. To elucidate functional properties of myocilin, recombinant myocilin was expressed in 293 EBNA cells and purified by Ni-chelate and heparin chromatography. Culture plates were coated with myocilin, and primary rat mesangial cells and cells from an immortal murine podocyte cell line were seeded onto the plates in serum free conditions. Both cell types showed concentration-dependant attachment to myocilin, an effect that was statistically significant and could be blocked with specific antibodies. When compared to equal amounts of fibronectin or collagen 1, myocilin was less effective in promoting substrate adhesion. Synergistic effects in substrate adhesion were observed when myocilin was added to low concentrations of fibronectin. Twenty-five percent of cells that had attached to myocilin substrates showed spreading and expressed focal contacts which were labeled by vinculin/phalloidin staining. Comparable findings were observed when human or murine trabecular meshwork cells were seeded on myocilin substrates. Adhesive properties of myocilin required multimer formation, and were not observed when culture plates were coated with a C-terminal fragment of myocilin, containing the olfactomedin domain. We conclude that myocilin promotes substrate adhesion of podocytes and mesangial cells, and might contribute to cell-matrix adhesion of both cell types in vivo.     

Enterocin 012, a bacteriocin produced by Enterococcus gallinarum isolated from the intestinal tract of ostrich

Enterococcus gallinarum strain 012, isolated from the duodenum of ostrich, produced enterocin 012 which is active against Ent. faecalis, Lactobacillus acidophilus, Lact. sake, Listeria innocua, Propionibacterium acidipropionici, Propionibacterium sp., Clostridium perfringens, Pseudomonas aeruginosa and Salmonella typhimurium. One of the four pathogenic strains of Escherichia coli isolated from the intestinal tract of ostrich was inhibited by enterocin 012. No antimicrobial activity was recorded against Bacillus cereus, Cl. sporogenes, Cl. tyrobutyricum, Leuconostoc cremoris, Pediococcus pentosaceus, Staphylococcus carnosus and Streptococcus thermophilus. Enterocin 012 was resistant to treatment with lysozyme, catalase, lipase and papain, but sensitive to Proteinase K, alpha-chymotrypsin, trypsin and pepsin. Treatment of enterocin 012 with gastric juice from the duodenum resulted in a 50% loss of antibacterial activity. Half of the activity was lost when incubated at 80 degrees C for 30 min, or when kept overnight at a pH of 1.0-5.0 and pH 11.0 and 12.0, respectively. Enterocin 012 production started in mid-logarithmic growth and reached a maximum of 800 AU ml-1, but increased further to 1600 AU ml-1 in the stationary growth phase. The peptide is approximately 3.4 kDa in size, as determined after partial purification with Amberlite XAD-1180 and ammonium sulphate precipitation, followed by tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The mechanism of antimicrobial activity against Lact. sake LMG 13558 is bactericidal and caused cell lysis of active growing cells.     

正常人肠道肠球菌耐药性与生物膜形成关系的初探

目的了解正常人肠道肠球菌对临床常用抗生素的耐药水平和其生物膜的形成情况,并初步探讨肠球菌的耐药性与其生物膜形成之间的关系。方法用K-B法测定正常人肠道肠球菌对15种抗生素的敏感性,用96孔聚苯乙烯板进行生物膜形成试验。结果生物膜形成阳性菌株对高浓度链霉素、四环素和红霉素的耐药性(耐药率分别为42.9%、90.5%、71.4%)显著高于生物膜形成阴性的菌株(耐药率分别为4.8%、38.1%、42.8%),对其余12种抗生素的耐药性与生物膜形成阴性株差异无统计学意义。结论生物膜形成对肠球菌耐药性增强有一定作用,但还与其本身耐药性和抗生素的性质有关。     

Identification and properties of an alpha-amylase from a strain of Eubacterium sp. isolated from the rat intestinal tract.

1. A bacterial amylase was isolated from the intestinal content of monoxenic rats inoculated with Eubacterium sp. B86. 2. Affinity chromatography on cross-linked starch allowed its separation from rat endogenous amylases. 3. The bacterial enzyme was characterized by its pI, molecular weight and action pattern. It behaves as a typical endo-amylase (alpha-amylase).     

Cardiac myofibroblasts isolated from the site of myocardial infarction express endothelin de novo

Recently it was demonstrated that treatment with a nonselective endothelin (ET) receptor antagonist significantly reduces myocardial infarct size, which suggests a major role for ET in tissue repair following myocardial infarction (MI). Tissue repair and remodeling found at the site of MI are mainly attributed to myofibroblasts (myoFbs), which are phenotypically transformed fibroblasts that express alpha-smooth muscle actin. It is unclear whether myoFbs generate ET peptides and consequentially regulate pathophysiological functions de novo through expression of the ET-1 precursor (prepro-ET-1), ET-converting enzyme-1 (ECE-1), a metalloprotease that is required to convert Big ET-1 to ET-1 and ET receptors. To address these intriguing questions, we used cultured myoFbs isolated from 4-wk-old MI scar tissue. In cultured cells, we found: 1) expression of mRNA for ET precursor gene (ppET1), ECE-1, and ETA and ETB receptors by semiquantitative RT-PCR; 2) phosphoramidon-sensitive ECE-1 activity, which converts Big ET-1 to biologically active peptide ET-1; 3) expression of ETA and ETB receptors; 4) elaboration of Big ET-1 and ET-1 peptides in myoFb culture media; and 5) upregulation of type I collagen gene expression and synthesis by ET, which was blocked by bosentan (a nonselective ETA- and ETB receptor blocker). These studies clearly indicated that myoFbs express and generate ET-1 and receptor-mediated modulation of type I collagen expression by ET-1. Locally generated ET-1 may contribute to tissue repair of the infarcted heart in an autocrine/paracrine manner.     

Biofilm formation by Acinetobacter baumannii strains isolated from urinary tract infection and urinary catheters

Systemic infections in avian species caused by avian pathogenic Escherichia coli (APEC) are economically devastating to poultry industries worldwide. To unravel factors possibly involved in APEC pathogenicity, suppression subtractive hybridization was applied, leading to the identification of a putative APEC autotransporter adhesin gene aatA in our previous study. In this study, pathogenic mechanism of AatA was further determined. A deletion mutant of aatA was constructed in the APEC DE205B, which results in the reduced capacity to adhere to DF-1 cells, defective virulence in vivo, and decreased colonization capacity in lung during the systemic infection compared with the wild-type strain. Furthermore, these capacities were restored in the complementation strains. These results indicated that AatA makes a significant contribution to APEC virulence through bacterial adherence to host tissues in vivo and in vitro. In addition, aggregation assays for strain AAEC189 expressing aatA indicated that AatA mediates cell aggregation and settling of cells. However, this cell aggregation is blocked by Type I fimbriae. This study illustrates the first examination of the role of AatA in aggregation and systemic infection.