Flow cytometric analysis of
            <i>Lactobacillus plantarum</i>
            to monitor lag times, cell divisionand injury

Ueckert, J.; von-Caron, G. Nebe; Bos, Ad P.; Steeg, P.F. ter

doi:10.1046/j.1472-765x.1997.00225.x

Cited by 72 publications

(53 citation statements)

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“…Discrimination between intact and permeable cells by fluorescent stains has been used in many studies on bacteria, including a few recent applications on lactic acid bacteria. Injury of Lactobacillus plantarum by nisin treatment was observed with PI and carboxyfluorescein succinimidylester using FCM (28). Furthermore, the membrane permeability of L. lactis was measured with PI and carboxyfluorescein diacetate (cFDA) using spectrofluorimetry and fluorescence microscopy in a study of viability after various stress treatments (6).…”

Section: Discussionmentioning

confidence: 99%

Fluorescent Method for Monitoring Cheese Starter Permeabilization and Lysis

Bunthof

Schalkwijk

Meijer

et al. 2001

Appl Environ Microbiol

106

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Section: Discussionmentioning

confidence: 99%

Fluorescent Method for Monitoring Cheese Starter Permeabilization and Lysis

Bunthof

Schalkwijk

Meijer

et al. 2001

Appl Environ Microbiol

106

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“…The flow cytometry profile showed that about 99% of the cells were labeled. A doubling in the cell concentration resulted in reduction of the median fluorescence intensity to half (9,17). Thus, the generation time (t d ) could be determined from the fluorescence intensity of the cells as t d ϭ ln2 ϫ (time interval)/(change in ln fluorescence intensity), where ln is the natural log value.…”

Section: Methodsmentioning

confidence: 99%

Permanent Colonization by Lactobacillus casei Is Hindered by the Low Rate of Cell Division in Mouse Gut

Lee

Low

et al. 2004

Appl Environ Microbiol

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Long residence times of probiotics in the intestinal tract would prolong their potential beneficial health effects and assist colonization. This study investigated the colonization potential of Lactobacillus casei Shirota in mouse intestine by using 5 (and 6)-carboxyfluorescein diacetate, succinimidyl ester (cFDA-SE) for assessment of doubling times in different parts of the intestine. The amounts of intestinal water overlying the surfaces of the duodenum, jejunum, ileum, and colon in BALB/c mice were 34.4 ؎ 2.9, 58.8 ؎ 6.8, 21.6 ؎ 2.2, and 8.0 ؎ 1.0 mg, respectively. Based on the residual concentrations of cFDA-SE-labeled lactobacilli on intestinal mucosal surfaces, the average half times for the wash-out of lactobacilli fed were estimated at 3.98, 1.55, 1.34, and 2.48 days in the duodenum, jejunum, ileum, and colon, respectively. The average doubling times of the lactobacilli, estimated from the residual fluorescent levels of surface-adhered cells, were 4.10, 4.78, 4.56, and 5.59 days in the duodenum, jejunum, ileum, and colon, respectively. It is estimated that the lactobacilli would have to achieve an average doubling time of 1.03 to 2.04 days to colonize the various sections of the mouse intestinal tract more permanently.Probiotic intestinal bacteria beneficially influence the health of the host by modulating the metabolic activities, immunity, and microbiota in the host's intestine (7,12). Lactobacilli have been used as antigen and cytokine delivery vehicles for oral immunization and disease treatment (11,16). Probiotic bacteria are selected for their beneficial health properties as well as their ability to tolerate intestinal conditions and achieve high growth rates in culture (13). However, no probiotic lactobacilli used in clinical trials and commercial production have been demonstrated to persist in fecal samples for more than a few weeks after their administration has been stopped (4,5,14,15). Such an effect is termed colonization resistance. The ability of exogenously administered probiotics to adhere to the mucosal cells and multiply in the intestinal tract has been questioned (2). There are recent reports on the recovery of consumed lactobacilli from human colonic biopsies after discontinuation of probiotic administration (1, 3, 18), thus providing direct evidence that probiotic lactobacilli are able to temporary colonize colonic mucosae. Prolonged adhesion and colonization of probiotic bacteria on intestinal mucosal surfaces could favor probiotic effects. The aim of this study was to understand the growth and colonization of lactobacilli in the intestinal tract, using the mouse as the model system. MATERIALS AND METHODSPreparation of fluorogenic dye. Five (and 6)-carboxyfluorescein diacetate, succinimidyl ester (cFDA-SE), is a nonfluorescent membrane-permeative ester which nonspecific prokaryotic and eukaryotic intracellular esterases convert to a fluorescent derivative that in turn is then covalently linked to intracellular proteins via the probe's succinimidyl group (19). cFDA-SE (2 mg) (Molecul...

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“…Intracellular Ca 21 levels were measured by flow cytometry as described (24). ZG cells were loaded with 2 mM Fluo-4-AM for Fig.…”

Section: Determination Of Intracellular Ca 21 Levelsmentioning

confidence: 99%

Sphingosine-1-phosphate stimulates aldosterone secretion through a mechanism involving the PI3K/PKB and MEK/ERK 1/2 pathways

Brizuela

Rábano

Gangoiti

et al. 2007

Journal of Lipid Research

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We reported recently that sphingosine-1-phosphate (S1P) is a novel regulator of aldosterone secretion in zona glomerulosa cells of adrenal glands and that phospholipase D (PLD) is implicated in this process. We now show that S1P causes the phosphorylation of protein kinase B (PKB) and extracellularly regulated kinases 1/2 (ERK 1/2), which is an indication of their activation, in these cells. These effects are probably mediated through the interaction of S1P with the Gi protein-coupled receptors S1P1/3, as pretreatment with pertussis toxin or with the S1P1/3 antagonist VPC 23019 completely abolished the phosphorylation of these kinases. Inhibitors of phosphatidylinositol 3-kinase (PI3K) or mitogen-activated protein kinase kinase (MEK) blocked S1P-stimulated aldosterone secretion. This inhibition was only partial when the cells were incubated independently with inhibitors of each pathway. However, aldosterone output was completely blocked when the cells were pretreated with LY 294002 and PD 98059 simultaneously. These inhibitors also blocked PLD activation, which indicates that this enzyme is downstream of PI3K and MEK in this system. We propose a working model for S1P in which stimulation of the PI3K/PKB and MEK/ERK pathways leads to the stimulation of PLD and aldosterone secretion.-Brizuela, L., M. Rábano, P. Gangoiti, N. Narbona, J. M. Macarulla, M. Trueba, and A. Gómez-Muñ oz. Sphingosine-1-phosphate stimulates aldosterone secretion through a mechanism involving the PI3K/PKB and MEK/ERK 1/2 pathways. J. Lipid Res.

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Flow cytometric analysis of Lactobacillus plantarum to monitor lag times, cell divisionand injury

Cited by 72 publications

References 1 publication

Fluorescent Method for Monitoring Cheese Starter Permeabilization and Lysis

Fluorescent Method for Monitoring Cheese Starter Permeabilization and Lysis

Permanent Colonization by Lactobacillus casei Is Hindered by the Low Rate of Cell Division in Mouse Gut

Sphingosine-1-phosphate stimulates aldosterone secretion through a mechanism involving the PI3K/PKB and MEK/ERK 1/2 pathways

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