scholarly journals Fluorescent Method for Monitoring Cheese Starter Permeabilization and Lysis

2001 ◽  
Vol 67 (9) ◽  
pp. 4264-4271 ◽  
Author(s):  
Christine J. Bunthof ◽  
Saskia van Schalkwijk ◽  
Wilco Meijer ◽  
Tjakko Abee ◽  
Jeroen Hugenholtz
Keyword(s):  
Nucleic Acid ◽  
Membrane Damage ◽  
Fluorescence Method ◽  
Epifluorescence Microscopy ◽  
Confocal Scanning Laser Microscopy ◽  
Acid Dye ◽  
High Concentration ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Syto 9

ABSTRACT A fluorescence method to monitor lysis of cheese starter bacteria using dual staining with the LIVE/DEAD BacLight bacterial viability kit is described. This kit combines membrane-permeant green fluorescent nucleic acid dye SYTO 9 and membrane-impermeant red fluorescent nucleic acid dye propidium iodide (PI), staining damaged membrane cells fluorescent red and intact cells fluorescent green. For evaluation of the fluorescence method, cells of Lactococcus lactis MG1363 were incubated under different conditions and subsequently labeled with SYTO 9 and PI and analyzed by flow cytometry and epifluorescence microscopy. Lysis was induced by treatment with cell wall-hydrolyzing enzyme mutanolysin. Cheese conditions were mimicked by incubating cells in a buffer with high protein, potassium, and magnesium, which stabilizes the cells. Under nonstabilizing conditions a high concentration of mutanolysin caused complete disruption of the cells. This resulted in a decrease in the total number of cells and release of cytoplasmic enzyme lactate dehydrogenase. In the stabilizing buffer, mutanolysin caused membrane damage as well but the cells disintegrated at a much lower rate. Stabilizing buffer supported permeabilized cells, as indicated by a high number of PI-labeled cells. In addition, permeable cells did not release intracellular aminopeptidase N, but increased enzyme activity was observed with the externally added and nonpermeable peptide substrate lysyl-p-nitroanilide. Finally, with these stains and confocal scanning laser microscopy the permeabilization of starter cells in cheese could be analyzed.

scholarly journals Trimeric G proteins regulate the cytosol-induced redistribution of Golgi enzymes into the endoplasmic reticulum

1995 ◽  
Vol 108 (4) ◽  
pp. 1805-1815 ◽  
Author(s):  
J. Hidalgo ◽  
M. Muniz ◽  
A. Velasco
Keyword(s):  
Endoplasmic Reticulum ◽  
G Proteins ◽  
Retrograde Transport ◽  
Microtubule Depolymerization ◽  
High Concentration ◽  
Intact Cells ◽  
Cytosolic Proteins ◽  
Permeabilized Cells ◽  
Integral Proteins ◽  
In Cells

Streptolysin O-permeabilized cells incubated with a high concentration (5-10 mg/ml) of cytosolic proteins and ATP-generating system exhibit redistribution into the endoplasmic reticulum (ER) of Golgi integral proteins (mannosidase II, galactosyltransferase, TGN 38), detected by immunofluorescence. In addition, mannosidase II is detected in the ER of cells exposed to a high concentration of cytosolic proteins and processed for immunolectron microscopy by immunoperoxidase. The redistribution process requires ATP and is not affected by previous microtubule depolymerization. Ultrastructural observations indicate that Golgi disassembly occurs by budding of coated vesicles. This stage of the process is inhibited by GTP-gamma S, AIF(3–5), transducin beta gamma subunits, and mastoparan, indicating the involvement of trimeric G proteins. At a later stage, vesicles lose their coats and fuse with the ER. This part of the process does not occur in cells incubated at either 15 degrees C or 20 degrees C, or exposed to N-ethylmaleimide. In cells treated with either cholera or pertussis toxin Golgi redistribution into the ER shows a 50-fold lower requirement for cytosolic factors than in untreated cells. These data suggest a regulatory role for both alpha s and alpha i trimeric G proteins in the normal Golgi-ER retrograde transport taking place in intact cells.

scholarly journals Using flow cytometry and light-induced fluorescence technique to characterizethe variability and characteristics of bioaerosols in springtime at Metro Atlanta, Georgia

2018 ◽  
Author(s):  
Arnaldo Negron ◽  
Natasha DeLeon-Rodriguez ◽  
Samantha M. Waters ◽  
Luke D. Ziemba ◽  
Bruce Anderson ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Nucleic Acid ◽  
Acid Content ◽  
Fungal Spores ◽  
Epifluorescence Microscopy ◽  
Nucleic Acid Content ◽  
Rain Event ◽  
Bacterial Cells ◽  
Sampling System ◽  
Spores And Pollen

Abstract. The abundance and speciation of primary biological aerosol particles (PBAP) is important for understanding their impacts on human health, cloud formation and ecosystems. Towards this, we have developed a protocol for quantifying PBAP collected from large volumes of air with a portable wet-walled cyclone bioaerosol sampler. A flow cytometry (FCM) protocol was then developed to quantify and characterize the PBAP populations from the sampler, which were confirmed against epifluorescence microscopy. The sampling system and FCM analysis were used to study PBAP in Atlanta, GA over a two-month period and showed clearly defined populations of DNA-containing particles: Low Nucleic Acid-content particles (bioLNA), High Nucleic Acid-content particles (HNA) being fungal spores and pollen. We find that daily-average springtime PBAP concentration (1 to 5 μm diameter) ranged between 1.4 × 104 and 1.1 × 105 m−3. The BioLNA population dominated PBAP during dry days (72 ± 18 %); HNA dominated the PBAP during humid days and following rain events, where HNA (e.g., wet-ejected fungal spores) comprised up to 92 % of the PBAP number. Concurrent measurements with a Wideband Integrated Bioaerosol Sensor (WIBS-4A) showed that FBAP and total FCM counts are similar; HNA (from FCM) significantly correlated with ABC type FBAP concentrations throughout the sampling period (and for the same particle size range, 1–5 μm diameter). However, the FCM bioLNA population, possibly containing bacterial cells, did not correlate to any FBAP type. The lack of correlation of any WIBS FBAP type with the bioLNA suggest bacterial cells may be more difficult to detect with autofluorescence than previously thought. Ιdentification of bacterial cells even in the FCM (bioLNA population) is challenging, given that the fluorescence level of stained cells at times may be comparable to that seen from abiotic particles. HNA and ABC displayed highest concentration on a humid and warm day after a rain event (4/14), suggesting that both populations correspond to wet-ejected fungal spores. Overall, information from both instruments combined reveals a highly dynamic airborne bioaerosol community over Atlanta, with a considerable presence of fungal spores during humid days, and a bioLNA population dominating bioaerosol community during dry days.

scholarly journals Analysis of mitochondrial morphology and function with novel fixable fluorescent stains.

1996 ◽  
Vol 44 (12) ◽  
pp. 1363-1372 ◽  
Author(s):  
M Poot ◽  
Y Z Zhang ◽  
J A Krämer ◽  
K S Wells ◽  
L J Jones ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Epifluorescence Microscopy ◽  
Mitochondrial Morphology ◽  
Microtubule Network ◽  
Multiple Features ◽  
Permeabilized Cells ◽  
Dye Staining ◽  
And Function

Investigation of mitochondrial morphology and function has been hampered because photostable, mitochondrion-specific stains that are retained in fixed, permeabilized cells have not been available. We found that in live cell preparations, the CMXRos and H2-CMXRos dyes were more photostable than rhodamine 123. In addition, fluorescence and morphology of mitochondria stained with the CMXRos and CMXRos-H2 dyes were preserved even after formaldehyde fixation and acetone permeabilization. Using epifluorescence microscopy, we showed that CMXRos and H2-CMXRos dye fluorescence fully co-localized with antibodies to subunit I of cytochrome c oxidase, indicating that the dyes specifically stain mitochondria. Confocal microscopy of these mitochondria yielded colored banding patterns, suggesting that these dyes and the mitochondrial enzyme localize to different suborganellar regions. Therefore, these stains provide powerful tools for detailed analysis of mitochondrial fine structure. We also used poisons that decrease mitochondrial membrane potential and an inhibitor of respiration complex II to show by flow cytometry that the fluorescence intensity of CMXRos and H2-CMXRos dye staining responds to changes in mitochondrial membrane potential and function. Hence, CMXRos has the potential to monitor changes in mitochondrial function. In addition, CMXRos staining was used in conjunction with spectrally distinct fluorescent probes for the cell nucleus and the microtubule network to concomitantly evaluate multiple features of cell morphology.

Immunocytochemical and radioimmunometrical demonstration of serotonin- and neuropeptide-immunoreactivities in the adult rat tapeworm,Hymenolepis diminuta(Cestoda, Cyclophyllidea)

Parasitology ◽  
1991 ◽  
Vol 103 (2) ◽  
pp. 275-289 ◽  
Author(s):  
D. M. McKay ◽  
I. Fairweather ◽  
C. F. Johnston ◽  
C. Shaw ◽  
D. W. Halton
Keyword(s):  
Nerve Cell ◽  
Peptide Yy ◽  
Hymenolepis Diminuta ◽  
Epifluorescence Microscopy ◽  
Confocal Scanning Laser Microscopy ◽  
Nerve Fibres ◽  
Cerebral Ganglia ◽  
Longitudinal Nerve ◽  
Small Nerve ◽  
Nerve Cords

Standard indirect immunocytochemical techniques have been interfaced with confocal scanning laser microscopy (for whole-mount preparations) and epifluorescence microscopy (for cryosections) to investigate the occurrence and distribution of serotoninergic and peptidergic nerve elements in adultH. diminuta. Serotonin (5-HT)-immunoreactivity (IR) was widespread throughout the worm, occurring in the paired cerebral ganglia, transverse commissure, the 10 longitudinal nerve cords and in a plethora of small nerve fibres of the peripheral nervous system. An abundance of serotoninergic nerve cell bodies was found in association with the lateral nerve cords. The genital atrium and accessory reproductive ducts were richly innervated with serotoninergic nerve fibres. Thirty-five antisera to 20 vertebrate regulatory peptides and 1 invertebrate peptide (FMRFamide) were used to screen the worm for neuropeptide IR. Immunostaining was obtained with antisera raised to pancreatic polypeptide (PP), peptide YY (PYY), neuropeptide Y (NPY), substance P (SP), peptide histidine isoleucine (PHI), xenopsin (XP) and FMRFamide. The most extensive pattern of IR occurred with antisera to PP and PYY, IR being evident in the cerebral ganglia, transverse commissure, longitudinal nerve cords and in small nerve fibres that ramified throughout the parenchyma. A series of bipolar nerve cell bodies between the median nerve cords displayed PP/PYY-IR. The distribution of FMRFamide-IR was reminiscent of the PP/PYY pattern but was less extensive. Comparison of the serotoninergic and peptidergic nervous systems has revealed general similarities and some distinct differences, especially with regard to the distribution of immunoreactive nerve cell bodies. Quantitative data are presented on the levels of PP-, SP-, PH1-, and gastrin-releasing peptide (GRP)-immunoreactivities demonstrable in acid-alcohol extracts of whole worms. The highest level of peptide IR determined was recorded for PP.

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 Glycerol Reduces Cross Hybridization on Nitrocellulose Membrane

2020 ◽  
Vol 38 (3) ◽  
pp. 199
Author(s):  
Narendra Yoga Hendarta ◽  
Abu Tholib Aman ◽  
Asmarani Kusumawati ◽  
Tri Wibawa
Keyword(s):  
Nucleic Acid ◽  
Point Of Care ◽  
Gc Content ◽  
Lateral Flow ◽  
Hybridization Signal ◽  
Nitrocellulose Membrane ◽  
High Concentration ◽  
Cross Hybridization ◽  
High Stringency ◽  
Stringency Condition

Lateral flow assay (LFD) based nucleic acid lateral flow (NALF)  method has been developed recently. The method met point of care testing (POCT) as simple and rapid procedures, less equipment, and can be performance by less skilled technician. NALF based on nucleic acid hybridizationis  more economical then immunochromatography assay which use antibody-antigen recognition. Cross hybridization has issued while used to differentiate organism with high GC content and high homology as high similarity genome. Some techniques has applied to give high stringency condition avoid cross hybridization reaction but need more procedure to apply. We found glycerol applied to buffer assay could reduce cross hybridization on nitrocellulose membrane. The study used 2 kinds of high stringency buffer as PBS and SSC bases and high concentration of ssDNA amplicon as sample. Without glycerol ingredient gave cross hybridization signal on test line. But used glycerol could reduce those even omitted with PBS based buffer assay. Beside those, glycerol could significantly increased hybridization signal in SSC based buffer assay (p<0.05).

scholarly journals Regulation of Ca2+ current in frog ventricular cardiomyocytes by guanosine 5'-triphosphate analogues and isoproterenol.

1993 ◽  
Vol 102 (3) ◽  
pp. 525-549 ◽  
Author(s):  
T D Parsons ◽  
H C Hartzell
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Ventricular Myocytes ◽  
Calcium Currents ◽  
Internal Perfusion ◽  
High Concentration ◽  
Patch Clamp Technique ◽  
Intact Cells ◽  
Molecule Interactions ◽  
Gamma S

Calcium currents (ICa) were measured in frog ventricular myocytes using the whole-cell patch clamp technique and a perfused pipette. To gain insight into the role of G proteins in the regulation of ICa in intact cells, the effect of internal perfusion with hydrolysis-resistant GTP analogues, guanylyl 5'-imidodiphosphate (GppNHp) or guanosine 5'-thiotriphosphate (GTP gamma S), on ICa stimulated by isoproterenol (Iso) or forskolin (Forsk) was examined. Significant differences were observed between the effects of the two GTP analogues. Internal perfusion of GppNHp resulted in a near-complete (approximately 80%) and irreversible inhibition of Iso-stimulated ICa. In contrast, internal perfusion with GTP gamma S resulted in only a partial (approximately 40%) inhibition of Iso- or Forsk-stimulated ICa. The fraction of the current not inhibited by GTP gamma S remained persistently elevated after the washout of Iso but declined to basal levels upon washout of Forsk. Excess internal GTP or GppNHp did not reduce the persistent ICa. Internal adenosine 5'-thiotriphosphate (ATP gamma S) mimicked the GTP gamma S-induced, persistent ICa. GppNHp sometimes induced a persistent ICa, but only if GppNHp was present at high concentration before Iso exposure. Inhibitors of protein kinase A inhibited both the GTP gamma S- and ATP gamma S-induced, persistent ICa. We conclude that: (a) GTP gamma S is less effective than GppNHp in inhibiting adenylyl cyclase (AC) via the inhibitory G protein, Gi; and (b) the persistent ICa results from a long-lived Gs-GTP gamma S complex that can activate AC in the absence of Iso. These results suggest that different hydrolysis-resistant nucleotide analogues may behave differently in activating G proteins and imply that the efficacy of G protein-effector molecule interactions can depend on the GTP analogue with which the G protein is activated.

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.

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