Evaluation of protein disulfide conversion in vitro using a continuous flow dialysis system

2013 ◽  
Vol 432 (2) ◽  
pp. 142-154 ◽  
Author(s):  
Xinzhao Grace Jiang ◽  
Tian Wang ◽  
Oliver Kaltenbrunner ◽  
Kenneth Chen ◽  
Gregory C. Flynn ◽  
...  
Keyword(s):  
Continuous Flow ◽  
Protein Disulfide ◽  
Flow Dialysis

Dynamic continuous-flow dialysis method to simulate intestinal digestion for in vitro estimation of mineral bioavailability of food

Talanta ◽  
2006 ◽  
Vol 68 (3) ◽  
pp. 549-557 ◽  
Author(s):  
Juwadee Shiowatana ◽  
Wutthika Kitthikhun ◽  
Upsorn Sottimai ◽  
Jeerawan Promchan ◽  
Kanokwan Kunajiraporn
Keyword(s):  
Continuous Flow ◽  
Mineral Bioavailability ◽  
Intestinal Digestion ◽  
Dialysis Method ◽  
Flow Dialysis

scholarly journals Impact of Chronic Tetracycline Exposure on Human Intestinal Microbiota in a Continuous Flow Bioreactor Model

Antibiotics ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 886
Author(s):  
Youngbeom Ahn ◽  
Ji Young Jung ◽  
Ohgew Kweon ◽  
Brian T. Veach ◽  
Sangeeta Khare ◽  
...  
Keyword(s):  
Molecular Analysis ◽  
Intestinal Microbiota ◽  
Continuous Flow ◽  
Antimicrobial Drug ◽  
Bioreactor Culture ◽  
Analysis Techniques ◽  
Human Intestinal Microbiota ◽  
Traditional Cultures ◽  
The Impact

Studying potential dietary exposure to antimicrobial drug residues via meat and dairy products is essential to ensure human health and consumer safety. When studying how antimicrobial residues in food impact the development of antimicrobial drug resistance and disrupt normal bacteria community structure in the intestine, there are diverse methodological challenges to overcome. In this study, traditional cultures and molecular analysis techniques were used to determine the effects of tetracycline at chronic subinhibitory exposure levels on human intestinal microbiota using an in vitro continuous flow bioreactor. Six bioreactor culture vessels containing human fecal suspensions were maintained at 37 °C for 7 days. After a steady state was achieved, the suspensions were dosed with 0, 0.015, 0.15, 1.5, 15, or 150 µg/mL tetracycline, respectively. Exposure to 150 µg/mL tetracycline resulted in a decrease of total anaerobic bacteria from 1.9 × 107 ± 0.3 × 107 down to 2 × 106 ± 0.8 × 106 CFU/mL. Dose-dependent effects of tetracycline were noted for perturbations of tetB and tetD gene expression and changes in acetate and propionate concentrations. Although no-observed-adverse-effect concentrations differed, depending on the traditional cultures and the molecular analysis techniques used, this in vitro continuous flow bioreactor study contributes to the knowledge base regarding the impact of chronic exposure of tetracycline on human intestinal microbiota.

scholarly journals Blastomyces Virulence Adhesin-1 Protein Binding to Glycosaminoglycans Is Enhanced by Protein Disulfide Isomerase

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Audrey Beaussart ◽  
Tristan Brandhorst ◽  
Yves F. Dufrêne ◽  
Bruce S. Klein
Keyword(s):  
Protein Disulfide Isomerase ◽  
Tandem Repeats ◽  
Pathogenic Fungi ◽  
Heparin Binding ◽  
Binding Studies ◽  
Disulfide Linkage ◽  
Disulfide Isomerase ◽  
Atomic Force ◽  
Protein Disulfide

ABSTRACT Blastomyces adhesin-1 (BAD-1) protein mediates the virulence of the yeast Blastomyces dermatitidis, in part by binding host lung tissue, the extracellular matrix, and cellular receptors via glycosaminoglycans (GAGs), such as heparan sulfate. The tandem repeats that make up over 90% of BAD-1 appear in their native state to be tightly folded into an inactive conformation, but recent work has shown that they become activated and adhesive upon reduction of a disulfide linkage. Here, atomic force microscopy (AFM) of a single BAD-1 molecule interacting with immobilized heparin revealed that binding is enhanced upon treatment with protein disulfide isomerase and dithiothreitol (PDI/DTT). PDI/DTT treatment of BAD-1 induced a plateau effect in atomic force signatures that was consistent with sequential rupture of tandem binding domains. Inhibition of PDI in murine macrophages blunted BAD-1 binding to heparin in vitro. Based on AFM, we found that a short Cardin-Weintraub sequence paired with a WxxWxxW sequence in the first, degenerate repeat at the N terminus of BAD-1 was sufficient to initiate heparin binding. Removal of half of the 41 BAD-1 tandem repeats led to weaker adhesion, illustrating their role in enhanced binding. Mass spectroscopy of the tandem repeat revealed that the PDI-induced interaction with heparin is characterized by ruptured disulfide bonds and that cysteine thiols remain reduced. Further binding studies showed direct involvement of thiols in heparin ligation. Thus, we propose that the N-terminal domain of BAD-1 governs the initial association with host GAGs and that proximity to GAG-associated host PDI catalyzes activation of additional binding motifs conserved within the tandem repeats, leading to enhanced avidity and availability of reduced thiols. IMPORTANCE Pathogenic fungi and other microbes must adhere to host tissue to initiate infection. Surface adhesins promote this event and may be required for disease pathogenesis. We studied a fungal adhesin essential for virulence (BAD-1; Blastomyces adhesin-1) and found that host products induce its structural reconfiguration and foster its optimal binding to tissue structures.

STUDIES ON ISOLATED RAT ADRENAL CELLS I. CONTINUOUS FLOW AND BATCH INCUBATIONS

1975 ◽  
Vol 78 (1) ◽  
pp. 110-121 ◽  
Author(s):  
H. E. Falke ◽  
H. J. Degenhart ◽  
G. J. A. Abeln ◽  
H. K. A. Visser ◽  
R. J. M. Croughs
Keyword(s):  
Production Rate ◽  
Continuous Flow ◽  
Response Curve ◽  
Steroid Biosynthesis ◽  
Adrenal Cells ◽  
Flow Apparatus ◽  
Lag Period ◽  
Number Of Cells ◽  
Isolated Rat

ABSTRACT A procedure for the continuous flow incubation of isolated adrenal cells is described. In this way the advantages of continuous flow incubations of adrenal tissue are combined with those of isolated adrenal cells. Suspensions of isolated adrenal cells were prepared by a modification of the collagenase method. A sigmoid dose-response curve was obtained when these cells were incubated with ACTH in batch incubations. Under these conditions (in the presence of 1 mU ACTH/ml) the corticosterone production rate remained constant during at least 240 min. This production rate was linearly related to the number of cells. Pre-incubation of the cells during 3 h resulted in an increased response to ACTH. In continuous flow incubations without ACTH the corticosterone production was negligible. With 100 μU ACTH/ml corticosterone production increased sharply after a short lag period. A maximum was reached after 60–75 min followed by a slow decrease. Cells pre-incubated in the continuous flow apparatus had a slightly diminished ACTH response without loss of affinity to ACTH. The continuous flow incubation of isolated adrenal cells offers new possibilities for the dynamic study of steroid biosynthesis in vitro. The method may also be valuable to study processes in a wide variety of other tissues.

Speed Modulation of the HeartWare HVAD to Assess In Vitro Hemocompatibility of Pulsatile and Continuous Flow Regimes in a Rotary Blood Pump

2018 ◽  
Vol 42 (9) ◽  
pp. 879-890 ◽  
Author(s):  
Jarod T. Horobin ◽  
Michael J. Simmonds ◽  
Deepika Nandakumar ◽  
Shaun D. Gregory ◽  
Geoff Tansley ◽  
...  
Keyword(s):  
Continuous Flow ◽  
Flow Regimes ◽  
Blood Pump ◽  
Rotary Blood Pump ◽  
Speed Modulation
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