scholarly journals Gelatinase Is Important for Translocation of Enterococcus faecalis across Polarized Human Enterocyte-Like T84 Cells

2005 ◽  
Vol 73 (3) ◽  
pp. 1606-1612 ◽  
Author(s):  
Jing Zeng ◽  
Fang Teng ◽  
Barbara E. Murray
Keyword(s):  
Enterococcus Faecalis ◽  
Regulatory System ◽  
Laboratory Strain ◽  
Human Colon ◽  
T84 Cells ◽  
Standard Assay ◽  
Fecal Isolate ◽  
Human Enterocyte ◽  
In Vitro Translocation

ABSTRACT Previously, in our laboratory, we established a two-chamber system to study translocation of Enterococcus faecalis across monolayers of polarized human colon carcinoma-derived T84 cells. By using the same system in the present study, we now show that disruption of gelE of strain OG1RF, which also has a polar effect on the cotranscribed sprE, as well as disruption of its regulatory system (fsrA, fsrB, and fsrC) resulted in a loss of detectable translocation by E. faecalis OG1RF; these mutants lost gelatinase (GelE) and serine protease (SprE) production by standard assay. A gelE deletion mutant of OG1RF (GelE− SprE+) also showed that significantly reduced translocation and complementation with the gelE gene (pTEX5438) in trans restored gelatinase and translocation, demonstrating that gelatinase is important for E. faecalis translocation. Complementation of fsrA, fsrB, and fsrC mutants with all three fsr genes also resulted in production of gelatinase and translocation. Furthermore, introduction of fsr genes into two non-gelatinase-producing E. faecalis isolates, the well-characterized laboratory strain JH2-2 and a human-derived fecal isolate, TX1322 (both of which have gelE but not fsrA or fsrB, are gelatinase negative, and do not translocate), resulted in gelatinase production by these strains and restored translocation across T84 monolayers, while transformation with pTEX5438 (gelE) showed little or no translocation and no detectable gelatinase, confirming the importance of both fsr and gelatinase for E. faecalis translocation. The importance of gelatinase production was also corroborated among 20 E. faecalis human isolates (7 fecal, 7 endocarditis, and 6 urine isolates), which showed translocation by all gelatinase-positive isolates but little to no translocation for gelatinase nonproducers. These results indicate that gelatinase is important for the successful in vitro translocation of E. faecalis across human enterocyte-like T84 cells.

scholarly journals Enterohemorrhagic Escherichia coli Colonization of Human Colonic EpitheliumIn VitroandEx Vivo

2014 ◽  
Vol 83 (3) ◽  
pp. 942-949 ◽  
Author(s):  
Steven B. Lewis ◽  
Vivienne Cook ◽  
Richard Tighe ◽  
Stephanie Schüller
Keyword(s):  
Escherichia Coli ◽  
Human Colon ◽  
Enterohemorrhagic Escherichia Coli ◽  
Care Needs ◽  
T84 Cells ◽  
Host Cell Membrane ◽  
Content Type ◽  
Colonic Biopsy

EnterohemorrhagicEscherichia coli(EHEC) is an important foodborne pathogen causing gastroenteritis and more severe complications, such as hemorrhagic colitis and hemolytic uremic syndrome. Pathology is most pronounced in the colon, but to date there is no direct clinical evidence showing EHEC binding to the colonic epithelium in patients. In this study, we investigated EHEC adherence to the human colon by usingin vitroorgan culture (IVOC) of colonic biopsy samples and polarized T84 colon carcinoma cells. We show for the first time that EHEC colonizes human colonic biopsy samples by forming typical attaching and effacing (A/E) lesions which are dependent on EHEC type III secretion (T3S) and binding of the outer membrane protein intimin to the translocated intimin receptor (Tir). A/E lesion formation was dependent on oxygen levels and suppressed under oxygen-rich culture conditions routinely used for IVOC. In contrast, EHEC adherence to polarized T84 cells occurred independently of T3S and intimin and did not involve Tir translocation into the host cell membrane. Colonization of neither biopsy samples nor T84 cells was significantly affected by expression of Shiga toxins. Our study suggests that EHEC colonizes and forms stable A/E lesions on the human colon, which are likely to contribute to intestinal pathology during infection. Furthermore, care needs to be taken when using cell culture models, as they might not reflect thein vivosituation.

Vancomycin-Dependent Enterococcus faecalis Clinical Isolates and Revertant Mutants

1999 ◽  
Vol 43 (1) ◽  
pp. 41-47 ◽  
Author(s):  
Françoise Van Bambeke ◽  
Murielle Chauvel ◽  
Peter E. Reynolds ◽  
Henry S. Fraimow ◽  
Patrice Courvalin
Keyword(s):  
Enterococcus Faecalis ◽  
Regulatory System ◽  
Clinical Isolates ◽  
Vancomycin Therapy ◽  
Gene Encoding ◽  
Vancomycin Resistant ◽  
Autophosphorylation Site ◽  
Rapid Emergence ◽  
Dipeptidase Activity

ABSTRACT Three vancomycin-dependent clinical isolates of Enterococcus faecalis of the VanB type were studied by determining (i) the sequence of the ddl gene encoding the hostd-Ala:d-Ala ligase and thevanS B-vanR B genes specifying the two-component regulatory system that activates transcription of the vanB operon, (ii) the level of expression of resistance genes by using dd-dipeptidase activity as a reporter, and (iii) the proportions of the peptidoglycan precursors synthesized. Each strain had a mutation in ddlleading to an amino acid substitution (D295 to V; T316 to I) or deletion (DAK251-253 to E) at invariant positions ind-Ala:d-Ala,d-Ala:d-Lac, andd-Ala:d-Ser ligases. These mutations resulted in impaired host d-Ala:d-Ala ligases since only precursors terminating in d-Ala-d-Lac were synthesized under vancomycin-inducing conditions. Two types of vancomycin-independent revertants of one isolate were obtained in vitro after growth in the absence of vancomycin: (i) vancomycin-resistant, teicoplanin-susceptible mutants had a 6-bp insertion in the hostddl gene, causing the E251-to-EYK change that restoredd-Ala:d-Ala ligase activity, (ii) constitutive vancomycin-resistant, teicoplanin-resistant mutants had substitutions (S232 to F or E247 to K) in the vicinity of the autophosphorylation site of the VanSB sensor and produced exclusively precursors ending in d-Ala-d-Lac. Vancomycin- and teicoplanin-dependent mutants obtained by growth in the presence of teicoplanin had an 18-bp deletion in VanSB, affecting residues 402 to 407 and overlapping the G2 ATP binding domain. The rapid emergence of vancomycin-independent revertants in vitro suggests that interruption of vancomycin therapy may not be sufficient to cure patients infected with vancomycin-dependent enterococci.

scholarly journals Regulation of Membrane Permeability by a Two-Component Regulatory System in Pseudomonas aeruginosa

2003 ◽  
Vol 47 (1) ◽  
pp. 95-101 ◽  
Author(s):  
Yanping Wang ◽  
Unhwan Ha ◽  
Lin Zeng ◽  
Shouguang Jin
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Multidrug Resistance ◽  
Membrane Permeability ◽  
Response Regulator ◽  
Regulatory System ◽  
Laboratory Strain ◽  
Clinical Isolates ◽  
Two Component ◽  
Increased Sensitivity

ABSTRACT Membrane impermeability is the major contributing factor to multidrug resistance in clinical isolates of Pseudomonas aeruginosa. By using laboratory strain PAK, a spontaneous P. aeruginosa mutant (mutant PAK1-3) whose membrane had reduced permeability and which displayed increased levels of resistance to various antibiotics, especially aminoglycosides, was isolated. By complementation of the mutant with a genomic clone library derived from wild-type strain PAK, a novel two-component regulatory system (PprA and PprB) was identified and was found to be able to increase the permeability of the bacterial membrane and render PAK1-3 sensitive to antibiotics. Furthermore, specific phosphorylation of the response regulator (PprB) by histidine kinase (PprA) was observed in vitro, demonstrating that they are cognate two-component regulatory genes. Introduction of a plasmid expressing the pprB gene into randomly chosen clinical isolates (n = 17) resulted in increased sensitivity to aminoglycosides in the majority of isolates (n = 13) tested. This is the first demonstration that P. aeruginosa membrane permeability can be regulated, providing an important clue in the understanding of the mechanism of membrane impermeability-mediated multidrug resistance in P. aeruginosa.

The influence of extracts from Rubus caesius leaves on human colon carcinoma cells cultured in vitro

Planta Medica ◽  
2014 ◽  
Vol 80 (16) ◽  
Author(s):  
R Paduch ◽  
M Tomczyk ◽  
A Wiater ◽  
A Dudek ◽  
M Pleszczynska ◽  
...  
Keyword(s):  
Colon Carcinoma ◽  
Human Colon ◽  
Carcinoma Cells ◽  
Colon Carcinoma Cells ◽  
Human Colon Carcinoma ◽  
Cells Cultured

In Vitro and in Vivo Comparison of Binding of 99m-Tc-Iabeled Anti-CEA MAb F33-104 with 99m-Tc-labeled Anti-CEA MAb BW431/26

1999 ◽  
Vol 38 (04) ◽  
pp. 115-119
Author(s):  
N. Oriuchi ◽  
S. Sugiyama ◽  
M. Kuroki ◽  
Y. Matsuoka ◽  
S. Tanada ◽  
...  
Keyword(s):  
Nude Mice ◽  
Binding Capacity ◽  
Colon Adenocarcinoma ◽  
Human Colon ◽  
Cell Binding ◽  
Vitro Binding ◽  
Labeling Method ◽  
Human Colon Adenocarcinoma

Summary Aim: The purpose of this study was to assess the potential for radioimmunodetection (RAID) of murine anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb) F33-104 labeled with technetium-99m (99m-Tc) by a reduction-mediated labeling method. Methods: The binding capacity of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA by means of in vitro procedures such as immunoradiometric assay and cell binding assay and the biodistribution of 99m-Tc-labeled anti-CEA MAb F33-104 in normal nude mice and nude mice bearing human colon adenocarcinoma LS180 tumor were investigated and compared with 99m-Tc-labeled anti-CEA MAb BW431/26. Results: The in vitro binding rate of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA in solution and attached to the cell membrane was significantly higher than 99m-Tclabeled anti-CEA MAb BW431/261 (31.4 ± 0.95% vs. 11.9 ± 0.55% at 100 ng/mL of soluble CEA, 83.5 ± 2.84% vs. 54.0 ± 2.54% at 107 of LS 180 cells). In vivo, accumulation of 99m-Tc-labeled anti-CEA MAb F33-104 was higher at 18 h postinjection than 99m-Tc-labeled anti-CEA MAb BW431/26 (20.1 ± 3.50% ID/g vs. 14.4 ± 3.30% ID/g). 99m-Tcactivity in the kidneys of nude mice bearing tumor was higher at 18 h postinjection than at 3 h (12.8 ± 2.10% ID/g vs. 8.01 ± 2.40% ID/g of 99m-Tc-labeled anti-CEA MAb F33-104, 10.7 ± 1.70% ID/g vs. 8.10 ± 1.75% ID/g of 99m-Tc-labeled anti-CEA MAb BW431/26). Conclusion: 99m-Tc-labeled anti-CEA MAb F33-104 is a potential novel agent for RAID of recurrent colorectal cancer.

Effect and mechanisms of action of the fixed combination STW 5 in secretion and motility of human colon in vitro.

Planta Medica ◽  
2007 ◽  
Vol 73 (09) ◽  
Author(s):  
D Krüger ◽  
S Wagner ◽  
CW Hann von Weyhern ◽  
F Zeller ◽  
O Kelber ◽  
...  
Keyword(s):  
Fixed Combination ◽  
Human Colon ◽  
Mechanisms Of Action

THYROID STIMULATORS AND THYROID STIMULATION

1971 ◽  
Vol 66 (3) ◽  
pp. 558-576 ◽  
Author(s):  
Gerald Burke
Keyword(s):  
Regulatory System ◽  
Control Site ◽  
Thyroid Cell ◽  
Primary Control ◽  
Physico Chemical ◽  
Site Of Action ◽  
Long Acting

ABSTRACT A long-acting thyroid stimulator (LATS), distinct from pituitary thyrotrophin (TSH), is found in the serum of some patients with Graves' disease. Despite the marked physico-chemical and immunologic differences between the two stimulators, both in vivo and in vitro studies indicate that LATS and TSH act on the same thyroidal site(s) and that such stimulation does not require penetration of the thyroid cell. Although resorption of colloid and secretion of thyroid hormone are early responses to both TSH and LATS, available evidence reveals no basic metabolic pathway which must be activated by these hormones in order for iodination reactions to occur. Cyclic 3′, 5′-AMP appears to mediate TSH and LATS effects on iodination reactions but the role of this compound in activating thyroidal intermediary metabolism is less clear. Based on the evidence reviewed herein, it is suggested that the primary site of action of thyroid stimulators is at the cell membrane and that beyond the(se) primary control site(s), there exists a multifaceted regulatory system for thyroid hormonogenesis and cell growth.

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