scholarly journals Development of a Flow Cytometric Method To Analyze Subpopulations of Bacteria in Probiotic Products and Dairy Starters

2002 ◽  
Vol 68 (6) ◽  
pp. 2934-2942 ◽  
Author(s):  
Christine J. Bunthof ◽  
Tjakko Abee
Keyword(s):  
Signal To Noise Ratio ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Flow Cytometric Method ◽  
Cell Counts ◽  
Plate Counts ◽  
Different Populations ◽  
Fcm Analysis ◽  
Dairy Fermentation ◽  
Fermentation Starters

ABSTRACT Flow cytometry (FCM) is a rapid and sensitive technique that can determine cell numbers and measure various physiological characteristics of individual cells by using appropriate fluorescent probes. Previously, we developed an FCM assay with the viability probes carboxyfluorescein diacetate (cFDA) and TOTO-1 {1′-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3dihydro(benzo-1,3-oxazole)-2-methylidene]-1-(3′-trimethylammoniumpropyl)-pyridinium tetraiodide} for (stressed) lactic acid bacteria (C. J. Bunthof, K. Bloemen, P. Breeuwer, F. M. Rombouts, and T. Abee, Appl. Environ. Microbiol. 67:2326-2335, 2001). cFDA stains intact cells with enzymatic activity, and TOTO-1 stains membrane-permeabilized cells. Here we used this assay to study the viability of bacterial suspensions in milk, dairy fermentation starters, and probiotic products. To facilitate FCM analysis of bacteria in milk, a commercially available milk-clearing solution was used. The procedure was optimized to increase the signal-to-noise ratio. FCM enumerations were accurate down to a concentration of 105 cells ml−1. The level of retrieval of Lactobacillus plantarum WCFS 1 suspended in milk was high, and viability was not affected by the procedure. The plate counts for cleared samples of untreated cell suspensions were nearly as high as the total FCM counts, and the correlation was strong (r > 0.99). In dairy fermentation starters and in probiotic products the FCM total cell counts were substantially higher than the numbers of CFU. Three functional populations could be distinguished: culturable cells, cells that are intact and metabolically active but not culturable, and permeabilized cells. The proportions of the populations differed in the products tested. This FCM method provides tools to assess the functionality of different populations in fermentation starters and probiotic products.

scholarly journals A permeabilized cell system identifies the endoplasmic reticulum as a site of protein degradation.

1991 ◽  
Vol 115 (5) ◽  
pp. 1225-1236 ◽  
Author(s):  
F J Stafford ◽  
J S Bonifacino
Keyword(s):  
Protein Degradation ◽  
Secretory Pathway ◽  
Fibroblast Cell ◽  
Cell System ◽  
Current Evidence ◽  
Permeabilized Cell ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Streptolysin O ◽  
A Site

Analysis of the fate of a variety of newly synthesized proteins in the secretory pathway has provided evidence for the existence of a novel protein degradation system distinct from that of the lysosome. Although current evidence suggests that proteins degraded by this system are localized to a pre-Golgi compartment before degradation, the site of proteolysis has not been determined. A permeabilized cell system was developed to examine whether degradation by this pathway required transport out of the ER, and to define the biochemical characteristics of this process. Studies were performed on fibroblast cell lines expressing proteins known to be sensitive substrates for this degradative process, such as the chimeric integral membrane proteins, Tac-TCR alpha and Tac-TCR beta. By immunofluorescence microscopy, these proteins were found to be localized to the ER. Treatment with cycloheximide resulted in the progressive disappearance of intracellular staining without change in the ER localization of the chimeric proteins. Cells permeabilized with the pore-forming toxin streptolysin O were able to degrade these newly synthesized proteins. The protein degradation seen in permeabilized cells was representative of that seen in intact cells, as judged by the similar speed of degradation, substrate selectivity, temperature dependence, and involvement of free sulfhydryl groups. Degradation of these proteins in permeabilized cells took place in the absence of transport between the ER and the Golgi system. Moreover, degradation occurred in the absence of added ATP or cytosol, and in the presence of apyrase, GTP gamma S, or EDTA; i.e., under conditions which prevent transport of proteins out of the ER. The efficiency and selectivity of degradation of newly synthesized proteins were also conserved in an isolated ER fraction. These data indicate that the machinery responsible for pre-Golgi degradation of newly synthesized proteins exists within the ER itself, and can operate independent of exogenously added ATP and cytosolic factors.

scholarly journals A new method for direct detection of the sites of actin polymerization in intact cells and its application to differentiated vascular smooth muscle

2010 ◽  
Vol 299 (5) ◽  
pp. C988-C993 ◽  
Author(s):  
Hak Rim Kim ◽  
Paul C. Leavis ◽  
Philip Graceffa ◽  
Cynthia Gallant ◽  
Kathleen G. Morgan
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Actin Polymerization ◽  
Direct Detection ◽  
New Method ◽  
Actin Dynamics ◽  
Tat Peptide ◽  
Isolated Cells ◽  
Intact Cells ◽  
Permeabilized Cells

Here we report and validate a new method, suitable broadly, for use in differentiated cells and tissues, for the direct visualization of actin polymerization under physiological conditions. We have designed and tested different versions of fluorescently labeled actin, reversibly attached to the protein transduction tag TAT, and have introduced this novel reagent into intact differentiated vascular smooth muscle cells (dVSMCs). A thiol-reactive version of the TAT peptide was synthesized by adding the amino acids glycine and cysteine to its NH2-terminus and forming a thionitrobenzoate adduct: viz. TAT-Cys-S-STNB. This peptide reacts readily with G-actin, and the complex is rapidly taken up by freshly enzymatically isolated dVSMC, as indicated by the fluorescence of a FITC tag on the TAT peptide. By comparing different versions of the construct, we determined that the optimal construct for biological applications is a nonfluorescently labeled TAT peptide conjugated to rhodamine-labeled actin. When TAT-Cys-S-STNB-tagged rhodamine actin (TSSAR) was added to live, freshly enzymatically isolated cells, we observed punctae of incorporated actin at the cortex of the cell. The punctae are indistinguishable from those we have previously reported to occur in the same cell type when rhodamine G-actin is added to permeabilized cells. Thus this new method allows the delivery of labeled G-actin into intact cells without disrupting the native state and will allow its further use to study the effect of physiological intracellular Ca2+ concentration transients and signal transduction on actin dynamics in intact cells.

Polyoma virus middle T antigen: relationship to cell membranes and apparent lack of ATP-binding activity

1982 ◽  
Vol 2 (10) ◽  
pp. 1187-1198 ◽  
Author(s):  
B S Schaffhausen ◽  
H Dorai ◽  
G Arakere ◽  
T L Benjamin
Keyword(s):  
Kinase Activity ◽  
T Antigen ◽  
Polyoma Virus ◽  
Intact Cells ◽  
Apparent Lack ◽  
Permeabilized Cells ◽  
Kinase Reaction ◽  
Middle T Antigen

Middle T antigen of polyoma virus is associated principally with the plasma membrane. Comparison of the trypsin sensitivity of middle T in intact cells and "inside out" membrane preparations showed that middle T is oriented towards the inside of the cell. This was confirmed by labeling of middle T in permeabilized cells, but not in intact cells, using [gamma-32P]ATP. Middle T molecules active in the in vitro kinase reaction could be differentiated from the bulk (metabolically labeled) middle T based on resistance to trypsin treatment. The active fraction also behaved differently from the bulk when cell frameworks were prepared with Triton-containing buffers; whereas the bulk middle T was evenly distributed in the soluble and cell framework fractions, the kinase-active forms were largely associated with the framework. Middle T molecules labeled in vivo with 32PO4 were found largely in the framework fraction, like the molecules that show kinase activity in vitro. Experiments with ATP affinity reagents 8-azido-ATP and 2,3-dialdehyde ATP have failed to label the middle T antigen. However, 2,3-dialdehyde ATP could be used to inhibit the kinase reaction. This raises the question of whether middle T antigen possesses intrinsic kinase activity or, rather, associates with a cellular tyrosine kinase.

Activation of G proteins may inhibit or stimulate surfactant secretion in rat alveolar type II cells

1994 ◽  
Vol 266 (4) ◽  
pp. L375-L381 ◽  
Author(s):  
M. S. Pian ◽  
L. G. Dobbs
Keyword(s):  
G Proteins ◽  
Tonic Inhibition ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Type Ii Cells ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Cyclic Monophosphate ◽  
Surfactant Secretion

To investigate how G proteins regulate surfactant secretion, we subjected rat alveolar type II cells to conditions known to activate or to inactivate G proteins. AlF-4, which activates G proteins, inhibited secretion in intact cells. Guanosine-5'-O-(3-thiotriphosphate), which activates G proteins in permeabilized cells, stimulated secretion at basal cytosolic [Ca2+], but inhibited secretion at higher [Ca2+]. In contrast, guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), which inactivates G proteins, stimulated secretion at each [Ca2+] tested. Because treatment with GDP beta S stimulated secretion at basal cytosolic [Ca2+], surfactant secretion appears to be subject to G protein-regulated tonic inhibition. Pertussis toxin (PTX) inhibited terbutaline- and ionomycin-stimulated secretion in intact cells, but did not inhibit secretion stimulated by either forskolin or 8-bromoadenosine 3',5'-cyclic monophosphate. Inhibition by PTX of terbutaline-stimulated, but not 8-bromoadenosine 3',5'-cyclic monophosphate- or forskolin-stimulated secretion, suggests that PTX-sensitive G proteins regulate beta-adrenergic-stimulated surfactant secretion proximal to second messenger generation. Inhibition of ionomycin-stimulated secretion, however, suggests that PTX-sensitive G proteins may also regulate non-receptor-mediated secretory events.

Control of the beat cycle of respiratory tract cilia by Ca2+ and cAMP

1992 ◽  
Vol 263 (2) ◽  
pp. L232-L242 ◽  
Author(s):  
A. B. Lansley ◽  
M. J. Sanderson ◽  
E. R. Dirksen
Keyword(s):  
Respiratory Tract ◽  
Beat Frequency ◽  
Ciliary Beat Frequency ◽  
Ciliated Cells ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Whole Cells ◽  
Phase Reduction ◽  
Before And After ◽  
Ciliary Beat

Beat frequency and the duration of the constituent recovery, effective, and rest phases of the beat cycle of respiratory tract cilia were measured photoelectronically before and after manipulation with ionomycin or isoproterenol. Both ionomycin, acting by increasing intracellular Ca2+, and isoproterenol, acting by elevating intracellular adenosine 3',5'-cyclic monophosphate (cAMP), increased beat frequency by reducing the duration of the three phases of the ciliary beat cycle in a similar manner. The addition of increasing concentrations of ATP to ciliated cells permeabilized by exposure to saponin caused a pattern of phase reduction indistinguishable from that observed in whole cells. The beat frequency of permeabilized cells was slower than that of whole cells and insensitive to changes in Ca2+ and cAMP. Ca2+ and cAMP may regulate ciliary beat frequency by acting at a common site within intact cells, possibly regulating the rate at which the axoneme can use ATP or the availability of ATP to the axoneme.

Sorting of metabolic pathway flux by the plasma membrane in cerebrovascular smooth muscle cells

2000 ◽  
Vol 278 (4) ◽  
pp. C803-C811 ◽  
Author(s):  
Pamela G. Lloyd ◽  
Christopher D. Hardin
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Major Product ◽  
Intact Cells ◽  
Glycolytic Intermediate ◽  
Cerebral Microvessels ◽  
Permeabilized Cells ◽  
Direct Contrast ◽  
Cerebrovascular Smooth Muscle Cells ◽  
Fructose 1,6 Bisphosphate

We used β-escin-permeabilized pig cerebral microvessels (PCMV) to study the organization of carbohydrate metabolism in the cytoplasm of vascular smooth muscle (VSM) cells. We have previously demonstrated (Lloyd PG and Hardin CD. Am J Physiol Cell Physiol 277: C1250–C1262, 1999) that intact PCMV metabolize the glycolytic intermediate [1-13C]fructose 1,6-bisphosphate (FBP) to [1-13C]glucose with negligible production of [3-13C]lactate, while simultaneously metabolizing [2-13C]glucose to [2-13C]lactate. Thus gluconeogenic and glycolytic intermediates do not mix freely in intact VSM cells (compartmentation). Permeabilized PCMV retained the ability to metabolize [2-13C]glucose to [2-13C]lactate and to metabolize [1-13C]FBP to [1-13C]glucose. The continued existence of glycolytic and gluconeogenic activity in permeabilized cells suggests that the intermediates of these pathways are channeled (directly transferred) between enzymes. Both glycolytic and gluconeogenic flux in permeabilized PCMV were sensitive to the presence of exogenous ATP and NAD. It was most interesting that a major product of [1-13C]FBP metabolism in permeabilized PCMV was [3-13C]lactate, in direct contrast to our previous findings in intact PCMV. Thus disruption of the plasma membrane altered the distribution of substrates between the glycolytic and gluconeogenic pathways. These data suggest that organization of the plasma membrane into distinct microdomains plays an important role in sorting intermediates between the glycolytic and gluconeogenic pathways in intact cells.

Characterization of monoclonal antibodies recognizing each extracellular loop domain of occludin

2019 ◽  
Vol 166 (4) ◽  
pp. 297-308 ◽  
Author(s):  
Yoshimi Shimizu ◽  
Yoshitaka Shirasago ◽  
Takeru Suzuki ◽  
Tomoyuki Hata ◽  
Masuo Kondoh ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Transmembrane Protein ◽  
Site Directed Mutagenesis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Intact Cells ◽  
Flow Cytometric ◽  
Junction Protein ◽  
Fcm Analysis

Abstract The tight junction protein occludin (OCLN) is a four-pass transmembrane protein with two extracellular loops (ELs), and also functions as a co-receptor for hepatitis C virus (HCV). Recently, we reported the establishment of monoclonal antibodies (mAbs) recognizing each intact EL domain of OCLN that can strongly prevent HCV infection in vitro and in vivo, and these mAbs were applicable for flow cytometric (FCM) analysis, immunocytochemistry (ICC) and cell-based enzyme-linked immunosorbent assay. In the present study, we further examined the application of these anti-OCLN mAbs and characterized their binding properties. All four mAbs were available for immunoprecipitation. The three first EL (EL1)-recognizing mAbs were applicable for immunoblotting, but the second EL (EL2)-recognizing one was not. Using site-directed mutagenesis, we also determined residues of OCLN critical for recognition by each mAb. Our findings showed that the small loop between two cysteines of the EL2 domain is essential for the binding to one EL2-recognizing mAb and that the recognition regions by three EL1-recognizing mAbs overlap, but are not the same sites of EL1. To obtain a deeper understanding of OCLN biology and its potential as a therapeutic target, specific mAbs to detect or target OCLN in intact cells should be powerful tools for future studies.

scholarly journals Ceramide transport from endoplasmic reticulum to Golgi apparatus is not vesicle-mediated

1998 ◽  
Vol 333 (3) ◽  
pp. 779-786 ◽  
Author(s):  
Jan Willem KOK ◽  
Teresa BABIA ◽  
Karin KLAPPE ◽  
Gustavo EGEA ◽  
Dick HOEKSTRA
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Incubation Temperature ◽  
De Novo ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Streptolysin O ◽  
Vesicular Stomatitis ◽  
Glycoprotein Transport

Ceramide (Cer) transfer from the endoplasmic reticulum (ER) to the Golgi apparatus was measured under conditions that block vesicle-mediated protein transfer. This was done either in intact cells by reducing the incubation temperature to 15 °C, or in streptolysin O-permeabilized cells by manipulating the intracellular environment. In both cases, Cer transfer was not inhibited, as demonstrated by the biosynthesis of ceramide monohexosides and sphingomyelin (SM) de novo from metabolically (with [14C]serine) labelled Cer. This assay is based on the knowledge that Cer is synthesized, starting from serine and palmitoyl-CoA, at the ER, whereas glycosphingolipids and SM are synthesized in the (early) Golgi apparatus. Formation of [14C]glycosphingolipids and [14C]SM was observed under conditions that block vesicle-mediated vesicular stomatitis virus glycoprotein transport. These results indicate that [14C]Cer is transferred from ER to Golgi by a non-vesicular mechanism.

scholarly journals Agonist-induced inhibition of phosphatidylserine synthesis is secondary to the emptying of intracellular Ca2+ stores in Jurkat T-cells

1992 ◽  
Vol 288 (3) ◽  
pp. 785-789 ◽  
Author(s):  
C Pelassy ◽  
J P Breittmayer ◽  
C Aussel
Keyword(s):  
T Cells ◽  
Exchange System ◽  
Jurkat T Cells ◽  
Experimental Conditions ◽  
Intact Cells ◽  
Base Exchange ◽  
Permeabilized Cells ◽  
Atpase Inhibitors ◽  
Storage Organelle ◽  
Intracellular Compartments

The biosynthesis of phosphatidylserine (PtdSer) by the serine base-exchange enzyme system, in Jurkat T-lymphocytes, was inhibited in intact cells maintained in low-Ca(2+)-containing buffer (< 10 microM-Ca2+) by using Ca2+ ionophores (A23187 or ionomycin). The rise in cytosolic Ca2+ concentration under these experimental conditions was only due to the release of Ca2+ from intracellular compartments, suggesting that the inhibition of PtdSer synthesis was correlated with the emptying of intracellular Ca2+ pools. This was further studied in saponin-permeabilized cells, in which PtdSer synthesis was found to be inhibited by EGTA, Ca2+ ionophores (A23187 or ionomycin) and Ca(2+)-ATPase inhibitors [thapsigargin or 2,5-di-(t-butyl)-benzohydroquinone]. Since Ca(2+)-ATPase inhibitors impaired refilling of the Ca2+ stores with Ca2+, and since in CD3-activated Jurkat T-cells the Ca2+ stores remained empty after 1 h of treatment with anti-CD3 monoclonal antibodies, we suggest that PtdSer synthesis is mainly regulated by the level of Ca2+ in the intracellular compartments and that the Ca(2+)-dependent serine base-exchange system responsible for PtdSer synthesis is probably located within or close to a Ca(2+)-storage organelle.

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