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1
Gatto, C., and M. A. Milanick. "Inhibition of the red blood cell calcium pump by eosin and other fluorescein analogues." American Journal of Physiology-Cell Physiology 264, no. 6 (1993): C1577—C1586. http://dx.doi.org/10.1152/ajpcell.1993.264.6.c1577.
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This paper addresses the mechanism of inhibition of the plasma membrane Ca pump by fluorescein analogues and their isothiocyanate derivatives. Eosin (i.e., tetrabromofluorescein) was found to be one of the most potent reversible inhibitors of the erythrocyte Ca pump [half-maximal inhibitory concentration (IC50) < 0.2 microM]; fluorescein itself was about four orders of magnitude less potent (IC50 approximately 1,000 microM). Eosin decreased the maximum influx and thus did not compete with ATP for the Ca pump. Irreversible inhibition produced by the isothiocyanate analogues of eosin and fluorescein [eosin 5-isothiocyanate (EITC) and fluorescein 5-isothiocyanate (FITC), respectively] was also studied. While EITC bound reversibly at the eosin site, two results suggest that EITC does not react covalently at this site: 1) eosin did not alter the time course of the EITC irreversible reaction, and 2) the concentration dependence for reversible EITC inhibition was different from the concentration dependence for irreversible EITC inhibition. ATP did slow the rate of inactivation of both EITC and FITC consistent with the idea that EITC and FITC bind to the ATP site. Our results are consistent with eosin and ATP binding to separate sites and EITC reacting covalently at the ATP site, but not the eosin site.
2
Tzeng, C. M., L. H. Hsu, and R. L. Pan. "Inhibition of tonoplast ATPase from etiolated mung bean seedlings by fluorescein 5′-isothiocyanate." Biochemical Journal 285, no. 3 (1992): 737–43. http://dx.doi.org/10.1042/bj2850737.
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Fluorescein 5′-isothiocyanate (FITC) was used to modify the lysine residue in the active site of tonoplast H(+)-ATPase from etiolated mung-bean (Vigna radiata L.) seedlings. FITC caused marked inactivation of the enzyme activities of both membrane-bound and soluble ATPase and its associated H+ translocation. The SDS/PAGE pattern revealed that the FITC-binding site was in the large (A) subunit of ATPase. Inhibition could be substantially prevented by its physiological substrate ATP, pyrophosphate and nucleotides in the decreasing order: ATP greater than pyrophosphate greater than ADP greater than AMP greater than GTP greater than CTP greater than UTP. The mode of inhibition by FITC was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first-order kinetics with a Ki of 0.33 mM, a minimum inactivation half-time of 110 s, and a first-order rate constant of 0.244 s-1. A double-logarithmic plot of apparent rate constant versus FITC concentration gave a slope of 0.913, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labelling studies indicated that the incorporation of approx. 1 mol of FITC/mol of ATPase is sufficient to inhibit ATPase completely. The enhancement and blue shift of emission maxima of FITC after modification of ATPase indicated that the labelled lysine residue was located in a relatively hydrophobic domain.
3
Chin, Suk Fun, Aressa Azman, Suh Cem Pang, and Sing Muk Ng. "Fluorescein-Labeled Starch Maleate Nanoparticles as Sensitive Fluorescent Sensing Probes for Metal Ions." Journal of Nanomaterials 2014 (2014): 1–8. http://dx.doi.org/10.1155/2014/108359.
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Fluorescein 5(6)-isothiocyanate starch maleate (FISM) nanoparticles were prepared by covalently attached fluorescein 5(6)-isothiocyanate (FITC) with starch maleate. FISM nanoparticles with a mean particle size of 87 nm were formed via self-assembly upon precipitation in ethanolic solution. FISM nanoparticles were strongly fluorescent with maximum emission wavelength of 518 nm. The fluorescence of FISM nanoparticles can be quenched by silver (Ag+) and lead (Pb2+) ions in a concentration dependent manner. We have demonstrated the first use of FISM nanoparticles as cheap and effective fluorescent sensing probes for Ag+and Pb2+ions with detection limits as low as 2.55×10−5 M and 3.64×10−5 M, respectively.
4
Hulspas, R., P. J. Krijtenburg, J. F. Keij, and J. G. Bauman. "Avidin-EITC: an alternative to avidin-FITC in confocal scanning laser microscopy." Journal of Histochemistry & Cytochemistry 41, no. 8 (1993): 1267–72. http://dx.doi.org/10.1177/41.8.7687265.
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Detection of fluorescein-5-isothiocyanate (FITC)-labeled conjugates is suboptimal in two-color confocal scanning laser microscopy (CLSM). This limits the detection of small, dimly fluorescent targets. We explored the possible advantages of applying eosin-5-isothiocyanate (EITC) conjugated to avidin (Av-EITC) as an alternative for Av-FITC in CSLM. Despite the lower quantum efficiency of EITC, we found that the measured Av-EITC and Av-FITC emission intensities were similar as a result of the standard filter combinations used for simultaneous two-color detection in the Bio-Rad MRC 600 CSLM. The advantage of Av-EITC was that its fading characteristics compared very favorably to those of Av-FITC. An excitation intensity-dependent increase in Av-EITC fluorescence was observed, followed by an exponential decrease. This increase in fluorescence allows longer observation times, averaging of several scans without loss of brightness, and thus detection of dimly fluorescent targets by CSLM.
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Hao, Yuzhi, Xiaoyan Yang, Yongzhong Shi, et al. "Magnetic gold nanoparticles as a vehicle for fluorescein isothiocyanate and DNA delivery into plant cells." Botany 91, no. 7 (2013): 457–66. http://dx.doi.org/10.1139/cjb-2012-0281.
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Magnetic gold nanoparticles (mGNPs) with uniform size and morphology synthesized by our sonication treatment method were covalently bound with fluorescein isothiocyanate (FITC) molecules. Driven by an external magnetic field, FITC-labelled nanoparticles were delivered into plant cells with and without cell walls, evident from sectional transmission electron microscopy images. Confocal images further indicate that the green fluorescence in canola protoplasts and walled cells indeed came from the FITC molecules, instead of the chloroplasts’ autofluorescence. FITC-labelled nanoparticles had a delivery efficiency of 95% based on confocal images. In further study, plasmids were covalently bound with mGNPs, and delivered into canola cells with and without cell walls. After culturing for 48 h followed by staining with 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid (X-Gluc), blue colour appeared in the protoplasts, while the walled canola cells showed a green colour that can be interpreted as the combination of blue and yellow from the suspension cells themselves. The presence of the blue colour indicates the expression of the GUS gene; therefore, the plasmids were successfully delivered into the canola cells. Furthermore, on examination, mGNPs were considered to be noncytotoxic by fluorescein diacetate staining.
6
Liu, Chao-Zong, Yi-Wen R. Wang, Ming-Ching Shen, and Tur-Fu Huang. "Analysis of Human Platelet Glycoprotein llb-llla by Fluorescein Isothiocyanate-Conjugated Disintegrins with Flow Cytometry." Thrombosis and Haemostasis 72, no. 06 (1994): 919–25. http://dx.doi.org/10.1055/s-0038-1648984.
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SummaryDisintegrins are a group of snake venom peptides which inhibit human platelet aggregation by acting as glycoprotein Ilb-IIIa (GPIIb-Ilia) antagonists. They are cysteine-rich, Arg-Gly-Asp (RGD)-containing peptides, and bind to GPIIb-Ilia complex on platelet membrane with a very high affinity (Kd, 10−7 ∼ 10−8 M). In this study, we analyzed GPIIb-Ilia complex on platelet membrane by flow cytometry using fluorescein isothiocyanate (FITC)-conjugated disintegrins as probes. Of these FITC-conjugated disintegrins, FITC-Rhodostomin is the most sensitive probe because Rhodostomin was conjugated with more FITC molecules than Trigramin and Halysin were. The binding fluorescence intensity of FITC-Trigramin (FITC-Tg), FITC-Halysin (FITC-Hy) and FITC-Rhodostomin (FITC-Rn) was measured in both resting and ADP-activated platelets of diluted human platelet-rich plasma. The binding fluorescence of FITC-disintegrins was abolished by EDTA and 7E3, a monoclonal antibody against GPIIb-Ilia. ADP markedly increased the fluorescence intensity of FITC-Tg and FITC-Hy bound on platelets especially when lower doses of these probes were used, whereas it had little effect on that of FITC-Rn. Therefore, FITC-Tg and FITC-Hy can be used for the detection of the activated platelets as noted by a higher ratio of fluorescence intensity (approx. 2-4) between ADP-activated and resting platelets as compared with that (approx. 1-1.3) in the case of FITC-Rn as the probe. The platelets from three patients with Glanzmann’s thrombasthenia were probed with FITC-disintegrins. As a result these three patients could be classified as type I thrombasthenia based on the extremely low level of GPIIb-Ilia detected by this method (<5% of normal value).
7
Fabiano, Angela, Elisa Brilli, Letizia Mattii, et al. "Ex Vivo and in Vivo Study of Sucrosomial® Iron Intestinal Absorption and Bioavailability." International Journal of Molecular Sciences 19, no. 9 (2018): 2722. http://dx.doi.org/10.3390/ijms19092722.
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The present study aimed to demonstrate that Sideral® RM (SRM, Sucrosomial® Raw Material Iron) is transported across the excised intestine via a biological mechanism, and to investigate the effect that this transport route may produce on oral iron absorption, which is expected to reduce the gastrointestinal (GI) side effects caused by the bioavailability of non-absorbed iron. Excised rat intestine was exposed to fluorescein isothiocyanate (FITC)-labeled SRM in Ussing chambers followed by confocal laser scanning microscopy to look for the presence of fluorescein-tagged vesicles of the FITC-labeled SRM. To identify FITC-labeled SRM internalizing cells, an immunofluorescence analysis for macrophages and M cells was performed using specific antibodies. Microscopy analysis revealed the presence of fluorescein positive particulate structures in tissues treated with FITC-labeled SRM. These structures do not disintegrate during transit, and concentrate in macrophage cells. Iron bioavailability was assessed by determining the time-course of Fe3+ plasma levels. As references, iron contents in liver, spleen, and bone marrow were determined in healthy rats treated by gavage with SRM or ferric pyrophosphate salt (FP). SRM significantly increased both area under the curve (AUC) and clearance maxima (Cmax) compared to FP, thus increasing iron bioavailability (AUCrel = 1.8). This led to increased iron availability in the bone marrow at 5 h after single dose gavage.
8
Harris, H. W., B. Botelho, M. L. Zeidel, and K. Strange. "Cytoplasmic dilution induces antidiuretic hormone water channel retrieval in toad urinary bladder." American Journal of Physiology-Renal Physiology 263, no. 1 (1992): F163—F170. http://dx.doi.org/10.1152/ajprenal.1992.263.1.f163.
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Antidiuretic hormone (ADH) increases the osmotic water permeability (Pf) of the toad urinary bladder by insertion of water channels into the apical cell membrane. Transepithelial water flow (Jv) reduces Pf by inducing endocytosis of apical water channels despite continuous ADH stimulation. This phenomenon is termed flux inhibition. We wished to determine whether cytoplasmic dilution or transcellular Jv causes flux inhibition because both have been proposed previously as a primary regulatory mechanism for this process. Apical membrane endocytosis was quantified by monitoring the uptake of the fluid phase marker fluorescein isothiocyanate dextran (FITC-dextran). FITC-dextran fluorescence was monitored in Triton X-100 extracts of epithelial cells as the ratio of total tissue fluorescence compared with background fluorescence. The background was defined as cellular autofluorescence and nonspecific tissue staining due to the presence of small amounts of free fluorescein contaminating the FITC-dextran. FITC-dextran uptake measured under symmetric isotonic (220 mosmol/kgH2O) conditions in either the absence (1.0 +/- 0.4 SD; n = 14) or presence (1.3 +/- 0.3; n = 4) of ADH was not statistically different from that of background. In contrast, flux inhibition induced by a 180 mosmol/kgH2O apical-to-basolateral osmotic gradient increased FITC-dextran uptake to 3.4 +/- 1.3 (n = 7). FITC-dextran uptake was identical in bladders exposed to symmetric hypotonic (150 mosmol/kgH2O) solutions during ADH (3.6 +/- 0.9; n = 6) or adenosine 3',5'-cyclic monophosphate (3.1 +/- 0.4 fold; n = 3) stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)
9
Murtazina, Dilyara A., Sergei P. Petukhov, Kenneth B. Storey, Alexander M. Rubtsov та Olga D. Lopina. "Phosphorylation of H,K-ATPase α-Subunit in Microsomes from Rabbit Gastric Mucosa by cAMP-Dependent Protein Kinase". Bioscience Reports 19, № 2 (1999): 109–14. http://dx.doi.org/10.1023/a:1020110510609.
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A 100-kDa protein that is a main component of the microsomal fraction from rabbit gastric mucosa is phosphorylated by cAMP-dependent protein kinase (PKA) in the presence of 0.2% Triton X-100. Microsomes from rabbit gastric mucosa possess activity of H,K-ATPase but not activity of Na,K-ATPase. Incubation of microsomes with 5 μM fluorescein 5′-isothiocyanate (FITC) results in both an inhibition of H,K-ATPase and labeling of a protein with an electrophoretic mobility corresponding to the mobility of the protein phosphorylated by PKA. The data suggest that the α-subunit of H,K-ATPase can be a potential target for PKA phosphorylation.
10
Liu, Chao-Zong, Buo-Tsang Hur, and Tur-Fu Huang. "Measurement of Glycoprotein llb/llla Blockade by Flow Cytometry with Fluorescein Isothiocyanate-conjugated Crotavirin, a Member of Disintegrins." Thrombosis and Haemostasis 76, no. 04 (1996): 585–91. http://dx.doi.org/10.1055/s-0038-1650626.
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SummaryThe blockade of platelet membrane glycoprotein IIb/IIIa by a monoclonal antibody, 7E3, was measured by flow cytometry using a fluorescein isothiocyanate-conjugated disintegrin, FITC-crotavirin, as the probe. After treatment of platelets with 7E3 or 7E3 F(ab’)2, there is a good correlation between the inhibition of platelet aggregation and the blockade of FITC-crotavirin’s binding to platelets. The content of glycoprotein IIb/IIIa for the subsequent binding of FITC-crotavirin to the 7E3-pretreated platelets highly correlated to the extent of glycoprotein Ilb/IIIa, remaining available. It was evidenced by the observation that the sum of glycoprotein Ilb/IIIa occupation by 7E3 and that of FITC-crotavirin approached the total amount of glycoprotein Ilb/IIIa expressed on the platelet membrane. This indicates that the percentage inhibition of FITC-crotavirin’s binding at the saturation dose reflects the extent of glycoprotein Ilb/IIIa blockade by 7E3. At the saturation binding concentration (5 |xg/ml), FITC-crotavirin did not displace platelet bound 7E3. Gating the light-scattering profile for platelets, the binding of FITC-crotavirin to platelet glycoprotein Ilb/IIIa could be easily determined in diluted whole blood by direct stain method. The available unoccupied glycoprotein Ilb/IIIa of platelets in the 7E3 or 7E3 F(ab’)2-pretreated whole blood were measured by flow cytometry at the saturation binding dose of FITC-crotavirin (4 fig/ml) and the data showed that the higher deconcentration of antibody added into whole blood, the lower debinding of FITC-crotavirin to platelets. This technique may provide an alternative rapid method for measuring the blockade of glycoprotein Ilb/IIIa by 7E3, a promising anti-thrombotic agent, thus providing a monitoring method for adjusting the therapeutic dose of 7E3 or its related derivatives.
11
Schreiber, R., and D. Häussinger. "Characterization of the swelling-induced alkalinization of endocytotic vesicles in fluorescein isothiocyanate-dextran-loaded rat hepatocytes." Biochemical Journal 309, no. 1 (1995): 19–24. http://dx.doi.org/10.1042/bj3090019.
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Short-term cultivated rat hepatocytes were allowed to endocytose fluorescein isothiocyanate (FITC)-coupled dextran and the apparent vesicular pH (pHves) was measured by single-cell fluorescence. After 2 h of exposure to FITC-dextran, the apparent pH in the vesicular compartments accessible to endocytosed FITC-dextran was 6.01 +/- 0.05 (n = 39) in normo-osmotic media. Hypo-osmotic exposure increased, whereas hyper-osmotic exposure decreased apparent pHves. by 0.18 +/- 0.02 (n = 26) and 0.12 +/- 0.01 (n = 23) respectively. Incubation of the cells with unlabelled dextran for 2h before a 2-h FITC-dextran exposure had no effect on apparent pHves and its osmosensitivity. When, however, hepatocytes were exposed to unlabelled dextran for 5 h after a 2 h exposure to FITC-dextran, in order to allow transport of endocytosed FITC-dextran to late endocytotic/lysosomal compartments, apparent pHves. decreased to 5.38 +/- 0.04 (n = 12) and the apparent pH in the vesicular compartment containing the dye was no longer sensitive to aniso-osmotic exposure. These findings indicate that the osomosensitivity of pHves. is apparently restricted to early endocytotic compartments. Aniso-osmotic regulation of apparent pHves. in freshly FITC-loaded hepatocytes was not accompanied by aniso-osmolarity-induced changes of the cytosolic free calcium concentration, and neither vasopressin nor extracellular ATP, which provoked a marked Ca2+ signal, affected apparent pHves. Dibutyryl-cyclic AMP (cAMP) or vanadate (0.5 mmol/l) were without effect on apparent pHves. and its osmosensitivity. However, pertussis toxin-treatment or genistein (but not daidzein) or the erbstatin analogue methyl 2,5-dihydroxycinnamate fully abolished the osmo-sensitivity of apparent pHves., but did not affect apparent pHves. It is concluded that regulation of pHves. by cell volume occurs in early endocytotic compartments, but probably not in lysosomes, and is mediated by a G-protein and tyrosine kinase-dependent, but Ca2+- and cAMP-independent mechanism.
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Legrand, D., J. Mazurier, P. Maes, E. Rochard, J. Montreuil, and G. Spik. "Inhibition of the specific binding of human lactotransferrin to human peripheral-blood phytohaemagglutinin-stimulated lymphocytes by fluorescein labelling and location of the binding site." Biochemical Journal 276, no. 3 (1991): 733–38. http://dx.doi.org/10.1042/bj2760733.
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Labelling of human lactotransferrin with fluorescein 5′-isothiocyanate (FITC) in an equimolar ratio inhibits the binding of the protein to phytohaemagglutinin-activated human peripheral-blood lymphocytes. Therefore it can be assumed that FITC reacts at, or near, the receptor-binding site. Three FITC-labelled peptides have been purified from a tryptic digest of the FITC-labelled lactotransferrin. The determination of their amino acid sequence and their localization on the primary structure of the protein permitted the identification of two FITC-accessible areas in the N-terminal lobe and one in the C-terminal lobe. In fact, only 10% of the total FITC was conjugated to one lysine residue (Lys579) of the C-terminal lobe, whereas most (80%) of the FITC was conjugated to three close lysine residues [Lys263 (65% of total fluorescence), Lys280 and Lys282 (15% of total fluorescence)] located in beta-turn structures, of the N-terminal domain I of human lactotransferrin. The results obtained show that the receptor-binding site should be located in the vicinity of the FITC-accessible Lys263, Lys280 and Lys282, and corroborate our preliminary results reporting the involvement of the N-terminal domain I in the binding of human lactotransferrin to mitogen-stimulated lymphocytes [Rochard, Legrand, Mazurier, Montreuil & Spik (1989) FEBS Lett. 255, 201-204]. In any case, FITC labelling is not suitable for studying the binding of lactotransferrin to activated lymphocytes and its use may lead to erroneous interpretations of cell binding experiments.
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Pataki, G., L. Czopf, T. Jilling, N. Marczin, J. Catravas, and S. Matalon. "Regulation of fluid-phase endocytosis in alveolar macrophages." American Journal of Physiology-Lung Cellular and Molecular Physiology 269, no. 4 (1995): L520—L526. http://dx.doi.org/10.1152/ajplung.1995.269.4.l520.
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We investigated whether fluid-phase endocytosis in rabbit alveolar macrophages (AM) was regulated by alterations in intracellular adenosine 3',5'-cyclic monophosphate (cAMP). Suspensions of freshly isolated AM were incubated with anionic dextrans (mol mass = 10 kDa), coupled to fluorescein isothiocyanate (FITC), at either 37 or 4 degrees C. There was a rapid increase in AM-associated fluorescence, quantified by laser flow-cytometry and video microscopy during the first hour of incubation at 37 degrees C, which was directly proportional to the amount of tracer present in the medium. In contrast, at 4 degrees C, AM fluorescence was similar to autofluorescence. Incubation of AM with forskolin (50 microM) or 3-isobutyl-1-methyl xanthine (IBMX; 0.1 mM) increased their cAMP content by 67 +/- 2 and 52 +/- 5% (mean +/- SE; n = 4) and decreased FITC-dextran uptake by 29 +/- 4 and 31 +/- 4% (n = 3). On the other hand, incubation of AM with 0.5 mM IBMX inhibited FITC-dextran uptake by 62 +/- 4% (n = 3), without any further increase in cAMP. Incubation of AM with 0.4 mM 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP), a cell-permeable analogue of cAMP, decreased FITC-dextran uptake by 48 +/- 5% (n = 6). Pulse-chase experiments showed that the rate of FITC-dextran exocytosis was not affected by cAMP. We concluded that fluid-phase endocytosis in rabbit AM is regulated by cAMP and by an additional, cAMP-independent mechanism of IBMX.
14
Hyslop, P. A., C. E. Kuhn, and R. D. Sauerheber. "Insulin stimulation of glucose transport in isolated rat adipocytes. Functional evidence for insulin activation of intrinsic transporter activity within the plasma membrane." Biochemical Journal 232, no. 1 (1985): 245–54. http://dx.doi.org/10.1042/bj2320245.
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We examined the effects of the membrane-impermeant amino-group-modifying agent fluorescein isothiocyanate (FITC) on the basal and insulin-stimulated hexose-transport activity of isolated rat adipocytes. Pre-treatment of cells with FITC causes irreversible inhibition of transport measured in subsequently washed cells. Transport activity was inhibited by approx. 50% with 2 mM-FITC in 8 min. The cells respond to insulin, after FITC treatment and removal, and the fold increase in transport above the basal value caused by maximal concentrations of insulin was independent of the concentration of FITC used for pre-treatment over the range 0-2 mM, where basal activity was progressively inhibited. The ability of FITC to modify selectively hexose transporters accessible only to the external milieu was evaluated by two methods. (1) Free intracellular FITC, and the distribution of FITC bound to cellular components, were assessed after dialysis of the homogenate and subcellular fractionation on sucrose gradients by direct spectroscopic measurement of fluorescein. Most (98%) of the FITC was associated with the non-diffusible fractions. Equilibrium sucrose-density-gradient centrifugation of the homogenate demonstrated that the subcellular distribution of the bound FITC correlated with the density distribution of a plasma-membrane marker, but not markers for Golgi, endoplasmic reticulum, mitochondria or protein. Exposing the cellular homogenate, rather than the intact cell preparation, to 2 mM-FITC resulted in a 4-5-fold increase in total bound FITC, and the density-distribution profile more closely resembled the distribution of total protein. (2) Incubation of hexokinase preparations with FITC rapidly and irreversibly inactivates this protein. However, both intracellular hexokinase total activity and its apparent Michaelis constant for glucose were unaffected in FITC-treated intact cells. Further control experiments demonstrated that FITC pre-treatment of cells had no effect on the intracellular ATP concentration or the dose-response curve of insulin stimulation of hexose transport. Since the fold increase of hexose transport induced by insulin is constant over the range of inhibition of surface-labelled hexose transporters, we suggest that insulin-induced insertion of additional transporters into the plasma membrane may not be the major locus of acceleration of hexose transport by the hormone.
15
Barrera, Gabriele, Loredana Serpe, Federica Celegato, et al. "Surface modification and cellular uptake evaluation of Au-coated Ni 80 Fe 20 nanodiscs for biomedical applications." Interface Focus 6, no. 6 (2016): 20160052. http://dx.doi.org/10.1098/rsfs.2016.0052.
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A nanofabrication technique based on self-assembling of polystyrene nanospheres is used to obtain magnetic Ni 80 Fe 20 nanoparticles with a disc shape. The free-standing nanodiscs (NDs) have diameter and thickness of about 630 nm and 30 nm, respectively. The versatility of fabrication technique allows one to cover the ND surface with a protective gold layer with a thickness of about 5 nm. Magnetization reversal has been studied by room-temperature hysteresis loop measurements in water-dispersed free-standing NDs. The reversal shows zero remanence, high susceptibility and nucleation/annihilation fields due to spin vortex formation. In order to investigate their potential use in biomedical applications, the effect of NDs coated with or without the protective gold layer on cell growth has been evaluated. A successful attempt to bind cysteine-fluorescein isothiocyanate (FITC) derivative to the gold surface of magnetic NDs has been exploited to verify the intracellular uptake of the NDs by cytofluorimetric analysis using the FITC conjugate.
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Velilla, Esther, Elisabet Rodríguez-Gonzalez, Francesca Vidal, and Maria-Teresa Paramio. "Microtubule and microfilament organization in immature, in vitro matured and in vitro fertilized prepubertal goat oocytes." Zygote 13, no. 2 (2005): 155–65. http://dx.doi.org/10.1017/s0967199405003229.
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The aim of our study was to analyse the cytoskeletal organization of prepubertal goat oocytes. Microtubule and microfilament organization during in vitro maturation of prepubertal and adult goat oocytes and presumptive zygotes of in vitro matured–in vitro fertilized (IVM-IVF) prepubertal goat oocytes were analysed. Oocytes were matured in M-199 with hormones and serum and inseminated with frozen-thawed spermatozoa. Oocytes and presumptive zygotes were treated with anti-α-tubulin antibody and fluorescein isothiocyanate (FITC)-labelled goat anti-mouse antibody to stain the microtubules. Microfilaments were localized by means of phalloidin 5 μg/ml conjugated with fluorescein isothiocyanate (FITC-phalloidin). DNA was stained with propidium iodide. Stained oocytes were observed under a confocal laser scanning microscope. At the germinal vesicle nuclear stage, microfilaments were distributed at the cortex of the oocytes. After in vitro maturation, 91.7% of metaphase II (MII) oocytes from adult goats displayed microfilaments in the cortex and within the polar body and were characterized by the presence of a microfilament thickening at the cortical region over the meiotic spindle. In prepubertal goat MII oocytes only 5.7% of oocytes displayed microfilaments at the cortex and within the polar body. After insemination, most of the zygotes displayed microfilaments distributed at the cortex. An undefined microtubular network was observed in adult and prepubertal goat oocytes at the germinal vesicle stage. After in vitro maturation, 100% of MII oocytes from adult goats displayed microtubules on the meiotic spindle and within the polar body. This pattern of distribution was observed in 71.6% of prepubertal goat oocytes. Undefined microtubule networks were present in most of the zygotes analysed. In conclusion, cytoskeletal differences were found between prepubertal and adult goat MII oocytes. Furthermore, most of the zygotes from IVM-IVF prepubertal goat oocytes displayed cytoskeletal anomalies.
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Pinard, Elisabeth, Nicolas Engrand, and Jacques Seylaz. "Dynamic Cerebral Microcirculatory Changes in Transient Forebrain Ischemia in Rats: Involvement of Type I Nitric Oxide Synthase." Journal of Cerebral Blood Flow & Metabolism 20, no. 12 (2000): 1648–58. http://dx.doi.org/10.1097/00004647-200012000-00004.
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The diameter of surface microvessels and the erythrocyte velocity and flux through intraparenchymal capillaries in the parietal cortex were measured during transient global cerebral ischemia and reperfusion using laser-scanning confocal fluorescence microscopy in anesthetized rats. The role of nitric oxide (NO) from neurons in the microcirculatory changes was also investigated using 7-nitro-indazole (7-NI, 25 mg/kg, IP). Wistar rats (4 per group) equipped with a closed cranial window were given fluorescein isothiocyanate (FITC)-Dextran and FITC-labeled erythrocytes intravenously to respectively visualize the microvessels and the erythrocytes in the capillaries. Experiments were videorecorded on-line. Forebrains were made ischemic for 15 minutes and then reperfused for 120 minutes under the microscope. Ischemia was associated with a flattened EEG, a low persistent blood flow, and a transient leakage of fluorescein across the arteriole wall. Unclamping the carotid arteries led to immediate high blood flow in the arterioles, but it was not until 5 minutes later that the arterioles dilated significantly (181% ± 27%) and erythrocyte velocity in the capillaries increased significantly (460% ± 263%). Neither nonperfused capillaries nor erythrocyte capillary recruitment occurred. 7-Nitro-indazole significantly reduced the arteriole dilatation and prevented the increase in erythrocyte velocity and flux through capillaries in early reperfusion. 7-Nitro-indazole had no influence on the fluorescein leakage. The current study suggests a partial role for NO released from neurons in the postischemic microcirculatory changes and provides new findings on the timing of arteriole dilatation and blood—brain barrier opening, and on erythrocyte capillary circulation in global ischemia.
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Kim, D., P. M. Armenante, and W. N. Duran. "Transient analysis of macromolecular transport across microvascular wall and into interstitium." American Journal of Physiology-Heart and Circulatory Physiology 265, no. 3 (1993): H993—H999. http://dx.doi.org/10.1152/ajpheart.1993.265.3.h993.
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We studied the dynamics of macromolecular transport across the microvascular wall in the hamster cheek pouch using intravital microscopy and digital video-image analysis. We used fluorescein isothiocyanate-dextrans of 70,000 and 150,000 Da (FITC-Dextran 70 and 150, respectively) as tracers. We applied our mathematical model and our in vivo calibration to determine the diffusion coefficient (D) and the average fluid velocity (V) in the microvascular wall and in the interstitium from the experimental data. The value of D for FITC-Dextran 70 was 0.90 +/- 0.04 x 10(-11) cm2/s in the wall and 1.29 +/- 0.05 x 10(-8) cm2/s in the interstitium. In both regions, V was 2.05 +/- 0.05 x 10(-8) cm/s. The transport parameters for FITC-Dextran 150 were 0.27 +/- 0.02 x 10(-11) cm2/s, 0.55 +/- 0.05 x 10(-8) cm2/s, and 1.71 +/- 0.48 x 10(-8) cm/s for D in the wall and interstitium and V, respectively. The topical application of either calcium ionophore A23187 (7 x 10(-7) M) or bradykinin (5 x 10(-7) M) increased D for FITC-Dextran 70 and 150 2-fold and V 10-fold relative to their control values. We used these values to quantify the relative importance of the diffusive and convective mechanisms in the total solute flux. Molecular diffusion dominates convective transport in both the microvascular wall and the interstitial space.
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Pinto, Ricardo J. B., Nicole S. Lameirinhas, Gabriela Guedes, et al. "Cellulose Nanocrystals/Chitosan-Based Nanosystems: Synthesis, Characterization, and Cellular Uptake on Breast Cancer Cells." Nanomaterials 11, no. 8 (2021): 2057. http://dx.doi.org/10.3390/nano11082057.
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Cellulose nanocrystals (CNCs) are elongated biobased nanostructures with unique characteristics that can be explored as nanosystems in cancer treatment. Herein, the synthesis, characterization, and cellular uptake on folate receptor (FR)-positive breast cancer cells of nanosystems based on CNCs and a chitosan (CS) derivative are investigated. The physical adsorption of the CS derivative, containing a targeting ligand (folic acid, FA) and an imaging agent (fluorescein isothiocyanate, FITC), on the surface of the CNCs was studied as an eco-friendly methodology to functionalize CNCs. The fluorescent CNCs/FA-CS-FITC nanosystems with a rod-like morphology showed good stability in simulated physiological and non-physiological conditions and non-cytotoxicity towards MDA-MB-231 breast cancer cells. These functionalized CNCs presented a concentration-dependent cellular internalization with a 5-fold increase in the fluorescence intensity for the nanosystem with the higher FA content. Furthermore, the exometabolic profile of the MDA-MB-231 cells exposed to the CNCs/FA-CS-FITC nanosystems disclosed a moderate impact on the cells’ metabolic activity, limited to decreased choline uptake and increased acetate release, which implies an anti-proliferative effect. The overall results demonstrate that the CNCs/FA-CS-FITC nanosystems, prepared by an eco-friendly approach, have a high affinity towards FR-positive cancer cells and thus might be applied as nanocarriers with imaging properties for active targeted therapy.
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Tomeo, A. C., and W. N. Duran. "Resistance and exchange microvessels are modulated by different PAF receptors." American Journal of Physiology-Heart and Circulatory Physiology 261, no. 5 (1991): H1648—H1652. http://dx.doi.org/10.1152/ajpheart.1991.261.5.h1648.
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Using several platelet activating factor (PAF) receptor antagonists, we investigated whether a differential receptor sensitivity to PAF exists between the arteriolar and venular segments of the microcirculation. The microvascular bed of the hamster cheek pouch was observed with intravital fluorescent television microscopy. Alterations in arteriolar diameter and in the clearance of fluorescein isothiocyanate-dextran (FITC-Dx 150; mol wt 150,000) were measured in response to PAF. Topical application of 10(-7) M PAF elicits a 10-fold increase in FITC-Dx 150 clearance (mean +/- SE = 9,172 +/- 1,289 nl.2 h-1.g-1) compared with control and vasoconstricts arterioles (20-40 microns) to approximately 50% their control diameters. Pretreatment with WEB 2086 (2 mg/kg iv) or SDZ 63-675 (10 mg/kg iv) blocks PAF-induced vasoconstriction and significantly attenuates the clearance of FITC-Dx 150 evoked by PAF, producing mean clearance values of 2,164 +/- 604 and 3,648 +/- 262 nl.2 h-1.g-1 respectively. L-659,989 (2 or 10 mg/kg iv) and SDZ 63-675 (5 mg/kg iv) abolished the vasoconstrictor response but not the postcapillary venular permeability response to PAF. These data suggests the presence of heterogeneous PAF receptors between the pre- and postcapillary segments of the microcirculation.
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Tamura, Chiharu, Makoto Sakurai, and Katsuya Kato. "Preparation of Calcium Phosphate – Peptide Composites with Highly pH-Sensitive Drug Release." Key Engineering Materials 529-530 (November 2012): 486–89. http://dx.doi.org/10.4028/www.scientific.net/kem.529-530.486.
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Calcium phosphate (CaP) material has been used successfully for protein delivery. In the present work, our aim is the preparation of CaP combined with peptide and protein, and is the evaluation of pH-sensitive drug release ability. Protein used as the model drug was fluorescein isothiocyanate-bovine serum albumin (FITC-BSA). All compounds synthesized were low crystalline hydroxyapatite (HAp). The result of zeta potential indicated that incorporation of peptide in HAp could be changed the surface charge of particle. The CaP-BSA and CaP-BSA-pLys [Poly-L-lysine (pLys) was added to CaP composite.] were-23.1 mV and-12.0 mV, respectively. The ratio of protein release and dissolved Ca ion were measured by soaking CaP-peptide composites under various pH (pH 7.4, pH 6 and pH 5) conditions. BSA was released from CaP-BSA-pLys at only pH 5, not at pH 7.4 and pH 6.
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Cong, X., Y. Zhang, Q. H. He, et al. "Endothelial Tight Junctions Are Opened in Cholinergic-Evoked Salivation In Vivo." Journal of Dental Research 96, no. 5 (2017): 562–70. http://dx.doi.org/10.1177/0022034516685048.
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Blood vessels provide the original supplies for the formation of primary saliva, which is regulated by the tight junctions (TJs) between endothelial cells. Previous studies have shown that blood flow increases with vasodilatation during cholinergic-evoked salivation. However, changes in vascular paracellular permeability and the role of endothelial TJs in salivation are unknown. Here, we established an in vivo paracellular permeability detection system and observed that the endothelial TJs were permeable to 4-kDa fluorescein isothiocyanate (FITC)–dextran while impermeable to 40- and 70-kDa FITC-dextran under an unstimulated condition in mouse submandibular glands (SMGs). Pilocarpine increased the flux of 4- and 40-kDa FITC-dextran out of blood vessels but did not affect 70-kDa FITC-dextran. Claudin 5, a TJ protein specifically localized in salivary endothelial cells, was redistributed from the apicolateral membranes to the lateral and basolateral membranes and cytoplasm in cholinergic-stimulated mouse SMGs and freshly cultured human SMG tissues. In the transplanted SMGs from epiphora patients, we found that claudin 5 was present in the basolateral membranes and cytoplasm, instead of the apical region in control SMGs. Moreover, the level of phospho–myosin light chain 2 increased within the blood vessels of the pilocarpine-stimulated mouse SMGs and transplanted human SMGs, while the downstream molecule F-actin was reorganized in the endothelial cells of the transplanted human SMGs. Taken together, our findings provide direct visual evidence that the opening of endothelial TJs and the redistribution of claudin 5 are essential events contributing to cholinergic-evoked salivation, thus enriching our understanding of the secretory mechanisms that link blood flow to primary saliva formation by regulating the endothelial paracellular permeability.
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Boom, A., B. Flamion, M. Abramow, and R. Beauwens. "Drugs activating G proteins disturb cycling of ADH-dependent water channels in toad urinary bladder." American Journal of Physiology-Cell Physiology 269, no. 2 (1995): C424—C434. http://dx.doi.org/10.1152/ajpcell.1995.269.2.c424.
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In the toad urinary bladder, antidiuretic hormone (ADH)-mediated changes in water permeability depend on exocytic insertion and endocytic retrieval of water channels into and from the apical membrane, respectively. Because GTP-binding proteins (G proteins) are well-recognized regulators of vesicular trafficking throughout the cell, we tested the hypothesis that drugs interfering with G protein would modify the hydrosmotic response to ADH and the ADH-regulated formation of endosomes, as assessed by luminal incorporation of a fluid-phase marker [fluorescein isothiocyanate (FITC)-dextran, 70 kDa]. Mastoparan (4 microM) and compound 48/80 (poly-p-methoxyphenylethylmethylamine; 50 micrograms/ml), added to the luminal side of the toad urinary bladder, as well as AlF3 added to the serosal side (400 microM), inhibited ADH- and 8-bromoadenosine 3',5'-cyclic monophosphate-induced transepithelial water flow by > 50% and simultaneously enhanced cellular incorporation of FITC-dextran by > 200%. The pattern of FITC-dextran uptake observed using fluorescence microscopy both in scraped cells and in the intact bladder was granular, suggesting fluid-phase endocytosis. Mastoparan and AlF3, which are both probes of G proteins, increased FITC-dextran uptake only in the presence of ADH and a transepithelial osmotic gradient, i.e., under conditions where water channel-carrying endosomes presumably cycle. Therefore, we suggest that the ADH-dependent cycling of water channels could be controlled by one or more G proteins associated with the apical membrane and/or the water channel-carrying vesicles.
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van Iwaarden, J. F., J. A. van Strijp, M. J. Ebskamp, A. C. Welmers, J. Verhoef, and L. M. van Golde. "Surfactant protein A is opsonin in phagocytosis of herpes simplex virus type 1 by rat alveolar macrophages." American Journal of Physiology-Lung Cellular and Molecular Physiology 261, no. 2 (1991): L204—L209. http://dx.doi.org/10.1152/ajplung.1991.261.2.l204.
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In the present study we used flow cytometry to investigate the phagocytosis of fluorescein isothiocyanate-labeled herpes simplex virus type 1 (FITC-HSV-1) by rat alveolar macrophages and the effects of surfactant protein A (SP-A) on this process. The phagocytosis of FITC-HSV-1 by alveolar macrophages, which was studied as a model for virus phagocytosis in general, was strongly enhanced in the presence of SP-A. The SP-A-mediated phagocytosis was time and concentration dependent, reaching a maximal level after 15 min of incubation and at an SP-A concentration of 5 micrograms/ml. Using a fluorescence quenching technique, we could show that at least 65% of the viruses were indeed internalized by the macrophages. The addition of SP-A to the system was sufficient for the phagocytosis of FITC-HSV-1 by the alveolar macrophages, suggesting that SP-A acts as an opsonin. This hypothesis was further strengthened by the observation that F(ab')2 fragments of immunoglobulin G directed against SP-A could abolish FITC-HSV-1 phagocytosis by alveolar macrophages preincubated with SP-A. Comparing the opsonic capacity of serum and SP-A, SP-A proved to be twice as potent as serum in stimulating phagocytosis of FITC-HSV-1 by alveolar macrophages. Complement factor C1q, which is known to possess a similar collagen-like domain as SP-A, did not stimulate phagocytosis of FITC-HSV-1 by alveolar macrophages nor did it inhibit SP-A-mediated HSV-1 phagocytosis. This study demonstrates that SP-A may play an important role in the antiviral defenses of the lung.
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Voicu, Sorina Nicoleta, Miruna-Silvia Stan, Ionela Cristina Nica, et al. "Biocompatibility and Cellular Uptake of Fluorescent Chitosan Nanohydrogels in Murine Macrophages and B Lymphocytes." Materials Proceedings 4, no. 1 (2020): 10. http://dx.doi.org/10.3390/iocn2020-07851.
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Due to their intrinsic viscosity and hydrophilicity, nanohydrogel systems are used to significantly increase the efficiency of commercial contrast agents for MRI and thus effectively improve the sensitivity of the MRI technique. Since chitosan (CS) is a biocompatible polysaccharide frequently used in biomedical applications, we aimed to prepare chitosan nanohydrogels (NGs) by ionic gelation, the polysaccharide being further grafted with rhodamine (RBITC) and fluorescein isothiocyanate (FITC). In this way, the cytotoxic effect of different concentrations (5, 15, 30, 60, and 120 µg/mL) of the fluorescent CS-FITC and CS-RBITC NGs was investigated by assessing the plasma membrane integrity and the metabolic activity of RAW 264.7 murine macrophages and A20 mouse lymphoma B cells following exposure for 6 and 24 h. The cell viability (MTT assay) and lactate dehydrogenase activity were analyzed by spectrophotometric methods, while cellular uptake was observed by fluorescence microscopy. Our results showed that the exposure to CS-FITC and CS-RBITC NGs for 6 and 24 h did not induce significant changes to RAW 264.7 and A20 cells compared to control, proving a good nanogel biocompatibility for both cell lines. In addition, the fluorescence microscopy showed that cellular uptake was quite rapid and efficient for the NGs tested. Taking all of these into consideration, we can conclude that all types of nanohydrogels were biocompatible, being internalized in both cell types with predominantly cytoplasmic localization.
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Wang, Huiyang, Wenxiu Ding, Lijun Peng, et al. "Gadolinium-Loaded Solid Lipid Nanoparticles for Colorectal Tumor in MR Colonography." Journal of Biomedical Nanotechnology 16, no. 5 (2020): 594–602. http://dx.doi.org/10.1166/jbn.2020.2922.
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The current study aims to investigate the possibility of using solid lipid nanoparticles (SLNs)-enhanced magnetic resonance (MR) colonography to diagnose colorectal cancer. Gd-FITC-SLNs were synthesized by loading gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) and fluorescein isothiocyanate (FITC) simultaneously. Twenty mice received azoxymethane/dextran sulfate sodium (AOM/DSS) to induce adenocarcinoma of the colon and were divided into 4 groups, and 5 in per group. MR colonography were performed at different time periods before and after enema or intravenous injection of Gd-FITC-SLNs or Gd-DTPA. The results demonstrated SNR (signal-to-noise ratio) significantly increased from 1.56- to 1.76-fold within the colorectal tumors after the enema of Gd-FITC-SLNs (p < 0.001). No differences in SNR were observed after the enema of Gd-DTPA (p > 0.05). Besides, SNR increased from 1.54- to 1.72-fold within the colorectal tumors after the intravenous injection of Gd-FITC-SLNs (p < 0.001) while SNR increased from 1.39to 1.57-fold within the colorectal tumors after the injection of Gd-DTPA (p < 0.001). In addition, SNR within colorectal tumors significantly increased ranging from 20th to 140th min, and lasted for about 120 min (p < 0.05) after the enema of Gd-FITC-SLNs and SNR within colorectal tumors also significantly increased ranging from 0th hour to 8th hour, lasted for about 8 hour (p < 0.05) after the injection of Gd-FITC-SLNs. However, after the injection of Gd-DTPA, SNR within colorectal tumors significantly increased only ranging from 0th min to 20th min after administration (p < 0.01). Furthermore, hematoxylin and eosin (H&E) staining revealed that all mice developed adenocarcinoma of the colon. In summary, it is feasible by using Gd-FITC-SLNs in MR colonography to diagnose colorectal cancer. Enema of Gd-FITC-SLNs can provide marked enhancement of colorectal tumors quickly, and safer while intravenous injection of Gd-FITC-SLNs can provide a long-lasting enhancement of colorectal tumors in MR colonography. These findings present a potential clinical application of Gd-FITC-SLNs on MR colonography.
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Jin, Zhuqing, Jian Liang, Jiaqi Li, and Pappachan E. Kolattukudy. "Absence of MCP-induced Protein 1 Enhances Blood–Brain Barrier Breakdown after Experimental Stroke in Mice." International Journal of Molecular Sciences 20, no. 13 (2019): 3214. http://dx.doi.org/10.3390/ijms20133214.
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Focal cerebral ischemia can cause blood–brain barrier (BBB) breakdown, which is implicated in neuroinflammation and progression of brain damage. Monocyte chemotactic protein 1–induced protein 1 (MCPIP1) is a newly identified zinc-finger protein that negatively regulates inflammatory signaling pathways. We aimed to evaluate the impact of genetic MCPIP1 deletion on BBB breakdown and expression of BBB-related matrix metalloproteinases (MMPs) and tight junction proteins after cerebral ischemia/reperfusion (I/R) using MCPIP1-deficient (MCPIP1–/–) mice. Transient middle cerebral artery occlusion was induced in the MCPIP1–/– mice and their wild-type littermates for 2 h followed by reperfusion for 24 h. The degree of BBB breakdown was evaluated by injection of fluorescein isothiocyanate (FITC)-dextran. Quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry were performed to compare the expression of MMPs and claudin-5 and zonula occludens-1 (ZO-1). MCPIP1 deficiency in mice resulted in enhanced leakage of FITC-dextran, increased expression of MMP-9/3, and reduced expression of claudin-5 and ZO-1 in the brain compared to that seen in their wild-type littermates subjected to cerebral I/R. These results demonstrate that absence of MCPIP1 exacerbates cerebral I/R-induced BBB disruption by enhancing the expression of MMP-9/3 and the degradation of claudin-5 and ZO-1, providing novel insights into the mechanisms underlying BBB breakdown after cerebral ischemia/reperfusion
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Lee, Lai Yoke, Say Leong Ong, Jiang Yong Hu, et al. "Use of Semiconductor Quantum Dots for Photostable Immunofluorescence Labeling of Cryptosporidium parvum." Applied and Environmental Microbiology 70, no. 10 (2004): 5732–36. http://dx.doi.org/10.1128/aem.70.10.5732-5736.2004.
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ABSTRACT Cryptosporidium parvum is a waterborne pathogen that poses potential risk to drinking water consumers. The detection of Cryptosporidium oocysts, its transmissive stage, is used in the latest U.S. Environmental Protection Agency method 1622, which utilizes organic fluorophores such as fluorescein isothiocyanate (FITC) to label the oocysts by conjugation with anti-Cryptosporidium sp. monoclonal antibody (MAb). However, FITC exhibits low resistance to photodegradation. This property will inevitably limit the detection accuracy after a short period of continuous illumination. In view of this, the use of inorganic fluorophores, such as quantum dot (QD), which has a high photobleaching threshold, in place of the organic fluorophores could potentially enhance oocyst detection. In this study, QD605-streptavidin together with biotinylated MAb was used for C. parvum oocyst detection. The C. parvum oocyst detection sensitivity increased when the QD605-streptavidin concentration was increased from 5 to 15 nM and eventually leveled off at a saturation concentration of 20 nM and above. The minimum QD605-streptavidin saturation concentration for detecting up to 4,495 ± 501 oocysts (mean ± standard deviation) was determined to be 20 nM. The difference in the enumeration between 20 nM QD605-streptavidin with biotinylated MAb and FITC-MAb was insignificant (P > 0.126) when various C. parvum oocyst concentrations were used. The QD605 was highly photostable while the FITC intensity decreased to 19.5% ± 5.6% of its initial intensity after 5 min of continuous illumination. The QD605-based technique was also shown to be sensitive for oocyst detection in reservoir water. This observation showed that the QD method developed in this study was able to provide a sensitive technique for detecting C. parvum oocysts with the advantage of having a high photobleaching threshold.
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Mahdi, Fakhri, Zia Shariat Madar, Carlos D. Figueroa, and Alvin H. Schmaier. "Factor XII interacts with the multiprotein assembly of urokinase plasminogen activator receptor, gC1qR, and cytokeratin 1 on endothelial cell membranes." Blood 99, no. 10 (2002): 3585–96. http://dx.doi.org/10.1182/blood.v99.10.3585.
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Investigations were performed to define the factor XII (FXII) binding site(s) on cultured endothelial cells (HUVECs). Biotin- or fluorescein isothiocyanate (FITC)–FXII in the presence of 10 μM Zn2+ specifically binds to HUVEC monolayers or cells in suspension. Collagen-stimulated platelets release sufficient Zn2+ to support FXII binding. On laser scanning confocal microscopy or electron microscopy, FITC-FXII or Nanogold-labeled FXII, respectively, specifically bind to HUVECs. Antibodies to gC1qR, urokinase plasminogen activator receptor (uPAR) and, to a lesser extent, cytokeratin 1 (CK1) block FXII binding to HUVECs as determined by flow cytometry and soluble or solid phase binding assays. FITC-FXII on endothelial cells colocalizes with gC1qR, uPAR and, to a lesser extent, CK1 antigen. Combined recombinant soluble uPAR and CK1 inhibit 80% FITC-FXII binding to HUVECs. Peptide Y(39)HKCTHKGR(47) (YHK9) from the N-terminal region of FXII and peptide H(479)KHGHGHGKHKNKGKKNGKH(498) from HK's domain 5 cell-binding site block FITC-FXII binding to HUVECs. Peptide YHK9 also inhibits FXIIa's activation of prekallikrein and FXI on HUVECs. These combined investigations indicate that FXII through a region on its fibronectin type II domain binds to the same multiprotein receptor complex that comprises the HK binding site of HUVECs. However, plasma concentrations of HK and vitronectin inhibit FXII binding to HUVECs 100% and 50%, respectively, and plasma albumin and other proteins prevent a sufficient level of free Zn2+ to be available to support FXII binding to HUVECs. Thus, physiologic FXII expression on HUVECs is secondary to HK binding and highly restricted in its ability to initiate prekallikrein or FXI activation.
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Brabec, Marianne, Daniela Schober, Ernst Wagner, et al. "Opening of Size-Selective Pores in Endosomes during Human Rhinovirus Serotype 2 In Vivo Uncoating Monitored by Single-Organelle Flow Analysis." Journal of Virology 79, no. 2 (2005): 1008–16. http://dx.doi.org/10.1128/jvi.79.2.1008-1016.2005.
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ABSTRACT The effect of virus uncoating on endosome integrity during the early steps in viral infection was investigated. Using fluid-phase uptake of 10- and 70-kDa dextrans labeled with a pH-dependent fluorophore (fluorescein isothiocyanate [FITC]) and a pH-independent fluorophore (cyanine 5 [Cy5]), we determined the pHs of labeled compartments in intact HeLa cells by fluorescence-activated cell sorting analysis. Subsequently, the number and pH of fluorescent endosomes in cell homogenates were determined by single-organelle flow analysis. Cointernalization of adenovirus and 70-kDa FITC- and Cy5-labeled dextran (FITC/Cy5-dextran) led to virus-induced endosomal rupture, resulting in the release of the marker from the low-pH environment into the neutral cytosol. Consequently, in the presence of adenovirus, the number of fluorescent endosomes was reduced by 40% compared to that in the control. When human rhinovirus serotype 2 (HRV2) was cointernalized with 10-and 70-kDa FITC/Cy5-dextrans, the 10-kDa dextran was released, whereas the 70-kDa dextran remained within the endosomes, which also maintained their low pH. These data demonstrate that pores are generated in the membrane during HRV2 uncoating and RNA penetration into the cytosol without gross damage of the endosomes; 10-kDa dextran can access the cytosol through these pores. Whereas rhinovirus-mediated pore formation was prevented by the vacuolar ATPase inhibitor bafilomycin A1, adenovirus-mediated endosomal rupture also occurred in the presence of the inhibitor. This finding is in keeping with the low-pH requirement of HRV2 infection; for adenovirus, no pH dependence for endosomal escape was found with this drug.
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Kabir, Khondkar Ehteshamul, Daizo Koga, Kotaro Konno, and Takuma Takanashi. "Fluorescein-5 isothiocyanate conjugated-chitin-binding domain probe (FITC-CBD)-coupled detection of chitin in the peritrophic membrane of Monochamus alternatus (Coleoptera: Cerambycidae)." Journal of Asia-Pacific Entomology 15, no. 3 (2012): 397–400. http://dx.doi.org/10.1016/j.aspen.2012.01.009.
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SCHREIBER, Rainer, Fan ZHANG, and Dieter HÄUSSINGER. "Regulation of vesicular pH in liver macrophages and parenchymal cells by ammonia and anisotonicity as assessed by fluorescein isothiocyanate-dextran fluorescence." Biochemical Journal 315, no. 2 (1996): 385–92. http://dx.doi.org/10.1042/bj3150385.
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Short-term-cultivated rat hepatocytes and Kupffer cells were allowed to endocytose fluorescein isothiocyanate (FITC)-coupled dextran, in order to study the effects of aniso-osmotic exposure and NH4Cl on apparent vesicular pH (pHves) by single-cell fluorescence. Following a 2 h loading period with FITC–dextran in normo-osmotic (305 mosmol/l) medium, the apparent pHves was 6.01±0.05 (n = 39) in parenchymal cells and 4.94±0.04 (n = 76) in Kupffer cells. Under these conditions pHves in parenchymal cells, but not in Kupffer cells, was sensitive to changes in ambient osmolarity. Inhibition of vacuolar H+-ATPase by concanamycin A did not affect the osmosensitivity of pHves in parenchymal cells. However, the effects of anisotonicity on pHves were largely abolished in the presence of 4,4´-di-isothiocyanato-stilbene-2,2´-disulphonic acid (DIDS) or when extracellular chloride was substituted for gluconate. In neither Kupffer cells, nor liver parenchymal cells did hypo-osmotic cell swelling cause an increase in intracellular Ca2+. With regard to vesicular acidification, the following differences were noted between parenchymal and Kupffer cells. (1) In Kupffer cells endocytosed FITC–dextran reached a strongly acidic compartment with a pH value of approx. 5 within 5 min, whereas it took 4–5 h in parenchymal cells. Modification of pHves by hypo-osmolarity in Kupffer cells was only observed in a short-lived ‘early’ compartment with a pH value of approx. 6. (2) In contrast to pHves in parenchymal cells, pHves in Kupffer cells was very sensitive towards alkalinization by NH4Cl: addition of NH4Cl at 1 or 10 mM increased apparent pHves by 0.80 or 1.46 in Kupffer cells, but only by 0.18 or 0.56 in parenchymal cells. The low ammonia sensitivity of pHves in parenchymal cells was observed not only in the less acidic (pH approx. 6) endocytotic compartment which is reached by FITC–dextran within 2 h, but also in the stronger acidic compartment (pH approx. 5) which is reached after 4–5 h. (3) NH4Cl had no effect on the osmosensitivity of pHves in parenchymal cells, whereas in Kupffer cells pHves became sensitive to anisotonicity when NH4Cl was present. Osmosensitivity of pHves in Kupffer cells under these conditions, however, was not affected by genistein, DIDS or colchicine, whereas these compounds abolished the osmosensitivity of pHves in parenchymal cells. It is suggested that regulation of pHves by cell volume in liver parenchymal cells involves changes of vesicular chloride conductance. In addition, there are marked differences between Kupffer and parenchymal cells with respect to vesicular ammonia permeability and the kinetics of endocytotic membrane flow and acidification.
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Peerce, B. E. "Molecular mechanism of two noncompetitive inhibitors of Na(+)-glucose cotransporter: comparison of DCCD and PCMB." American Journal of Physiology-Gastrointestinal and Liver Physiology 264, no. 2 (1993): G300—G305. http://dx.doi.org/10.1152/ajpgi.1993.264.2.g300.
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The effects of noncompetitive inhibitors of Na(+)-dependent glucose uptake, p-chloromercuribenzoate N,N'-dicyclohexylcarbodiimide (DCCD), on substrate-induced cotransporter conformational changes were examined using fluorescein isothiocyanate (FITC) and tryptophan fluorescence. p-chloromercuribenzoate (PCMB) inhibited both substrate-induced conformational changes with similar concentration required for 50% quenching/enhancement of tryptophan or FITC fluorescence. In contrast, DCCD inhibited the Na(+)-induced conformational change with an apparent concentration resulting in 50% inhibition (K0.5) of 18 microM and the glucose-induced conformational change with an apparent K0.5 of 5 microM. DCCD slightly increased the apparent K0.5 for the Na+ concentration required for Na(+)-induced conformational change. DCCD and PCMB altered tryptophan accessibility to quench reagents in all three conformations. Tryptophan residues on the PCMB-treated cotransporter were more Cs+ than I- sensitive in contrast to the unlabeled cotransporter. The PCMB-treated cotransporter had a reduced response to Na+, suggesting that the mode of PCMB inactivation of cotransporter activity resulted from conformational changes in the substrate-free cotransporter. DCCD had a smaller effect on cotransporter tryptophan quench reagent susceptibility. However, DCCD-labeled cotransporter was equally accessible to I- and Cs+, and the DCCD-labeled cotransporter did not respond to substrates. Loss of charge distribution around cotransporter tryptophans correlated with loss of substrate-induced conformational changes.
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Douros, Vivian, Thomas Podor, Stephen Shaughnessy, Jeffrey Weitz, and Edward Young. "Localization of heparin and low-molecular-weight heparin in the rat kidney." Thrombosis and Haemostasis 91, no. 05 (2004): 927–34. http://dx.doi.org/10.1160/th03-10-0645.
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SummaryUnfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) are cleared, at least in part, by the kidneys through a poorly understood process. This study was undertaken to explore the mechanism of renal clearance of these drugs. Rats were given fluorescein-5-isothiocyanate (FITC)-labeled UFH or LMWH intravenously. At intervals after injection, rats were euthanized and the kidneys were harvested and subjected to immunohistochemical analysis and fluorescence microscopy. Both UFH and LMWH were localized to renal tubular cells and no immunoperoxidase staining or fluorescence was detected in glomeruli. Autoradiography demonstrated similar intracellular distribution of radio-labeled UFH suggesting that this phenomenon is independent of the method used to label heparin. Fluoresence in the tubules increased as a function of time after UFH injection, but reached a plateau after LMWH injection suggesting that the rate of renal tubular uptake depends on the molecular size of the heparin. When administered prior to FITC-labeled UFH or LMWH, probenecid, a renal organic anion inhibitor, decreased the renal tubular uptake of the heparins, whereas cimetidine, a renal organic cation inhibitor, had no effect. These findings suggest that renal excretion of UFH and LMWH primarily reflects tubular uptake via an organic anion transport mechanism.
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da Silva, Zigomar, Andressa Pereira de Souza, José Rodrigo Claudio Pandolfi, Francisco Noé da Fonseca, Carlos André da Veiga Lima-Rosa, and Mariana Groke Marques. "Comparison between electroporation and polyfection in pig sperm: efficiency and cell viability implications." Zygote 26, no. 4 (2018): 286–93. http://dx.doi.org/10.1017/s0967199418000205.
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SummaryThe aim of this study was to optimize protocols for electroporation (EP) and polyfection (PLF) using polyethyleneimine (PEI) for pig sperm transfection and to determine which method was the most efficient. For EP standardization, different voltages, amounts and times of electric pulses were tested using propidium iodide (PI) as reporter. For PLF standardization, different concentrations of fluorescein isothiocyanate (FITC)-labelled PEI (PEI/FITC) were incubated with sperm for different periods of time. Flow cytometry was performed to evaluate the best protocol in terms of cell viability, including cytoplasmic membrane, acrosome, chromatin integrities and mitochondrial potential using the FITC probe, PI, acridine orange (AO) and JC1. Transfections with the plasmid pmhyGENIE-5 were carried out under optimum conditions for each procedure (EP: 500 volts, 500 μs and two pulses; PLF: PEI 0.5 mg/ml and incubation time 10 min). Transfection efficacy was assessed by fluorescence in situ hybridization (FISH). A lower transfection rate was observed for sperm in the control group (17.8%) compared with EP (36.7%), with PLF (76.8%) being the most efficient. These results suggest that the EP and PEI could be an efficient and low cost transfection method for swine sperm. Notably, treated cells showed higher plasmatic the membrane damage (PMD) and/or acrosome damage (AD) indexes, therefore the combination of this procedure with biotechniques that facilitate fecundation (i.e. in vitro fertilization or intracytoplasmic sperm injection) or even inclusion of antioxidant or anti-apoptotic drugs to improve spermatozoa viability would be important.
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Baldwin, A. L., and L. M. Wilson. "Stationary red blood cells induce a negative charge on mucosal capillary endothelium." American Journal of Physiology-Gastrointestinal and Liver Physiology 266, no. 4 (1994): G685—G694. http://dx.doi.org/10.1152/ajpgi.1994.266.4.g685.
Full textAbstract:
Previous studies on Wistar-Furth rat intestinal mucosal capillaries show a variation in extent of binding of cationized ferritin (CF) to the endothelial surface. Three possible causes of this variation were investigated: 1) location of capillaries along the intestine, 2) time after feeding, and 3) effect of short-term hemostasis. In studies 1 (5 rats) and 2 (22 rats), the intestinal circulation was perfused with CF and perfusion fixed for electron microscopy. Observation of capillaries demonstrated that variation in CF binding could not be explained by factors 1 or 2. In study 3, capillaries on the mucosal surface were viewed using epifluorescent microscopy and were perfused with fluorescein isothiocyanate (FITC)-labeled CF. Initial perfusion produced no binding, but perfusion after 2 or 10 min of stasis gave extensive binding (19 rats). Stasis with red blood cells in saline (6 rats) or with hemoglobin solution (6 rats) gave similar results, but stasis with saline, platelet-rich plasma, or red cell ghosts in saline did not produce binding (8, 7, and 5 rats, respectively). Hemoglobin injected into the circulation without stasis also caused binding of FITC-CF, but not if the nitric oxide (NO) donor, SIN-1, was injected simultaneously. Stasis with saline containing the NO inhibitor, NG-nitro-L-arginine methyl ester produced some binding of FITC-CF (5 rats). We conclude that intestinal mucosal capillaries do not express a net negative surface charge under brisk flow conditions, but charge is quickly generated after blood stasis. It is possible that when red blood cells are stationary, hemoglobin may trap NO that otherwise might neutralize the negatively charged groups.
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Schreiber, R., B. Stoll, F. Lang, and D. Häussinger. "Effects of aniso-osmolarity and hydroperoxides on intracellular pH in isolated rat hepatocytes as assessed by (2′,7′)-bis(carboxyethyl)-5(6)-carboxyfluorescein and fluorescein isothiocyanate-dextran fluorescence." Biochemical Journal 303, no. 1 (1994): 113–20. http://dx.doi.org/10.1042/bj3030113.
Full textAbstract:
Freshly isolated rat hepatocytes were plated for 4-6 h and either loaded with (2′,7)-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) or allowed to endocytose fluorescein isothiocyanate (FITC)-coupled dextran in order to study the effects of aniso-osmotic exposure and oxidative stress on cytosolic (pHcyt) and apparent vesicular pH (pHves) by single-cell fluorescence recordings. In the presence of normo-osmotic (305 mosmol/l) medium pHcyt was 7.23 +/- 0.03 (n = 108), whereas an apparent pH of 6.07 +/- 0.02 (n = 156) was found in the vesicular compartment accessible to endocytosed FITC-dextran. Substitution of 60 mM NaCl against 120 mM raffinose had no effect on pHcyt or apparent pHves, whereas addition of NH4Cl increased both pHcyt and apparent pHves. Hypo-osmotic cell swelling lowered pHcyt, whereas simultaneously apparent pHves increased. These effects were rapidly reversible upon re-institution of normo-osmotic media. Similarly, an increase of apparent pHves was observed when cell swelling was induced by Ba2+, glutamine or histidine. Conversely, hyperosmotic cell shrinkage due to addition of NaCl or raffinose led to a cytosolic alkalinization and a vesicular acidification. Both, H2O2 (0.2 mmol/l) and t-butyl-hydroperoxide (0.2 mmol/l) were without effect on pHcyt, but lowered apparent pHves by about 0.2 pH units. Ba2+ (1 mmol/l) diminished the acidifying effect of the hydroperoxides by about 50%. Pretreatment of the cells with colchicine, but not with lumicolchicine, largely abolished the effects of aniso-osmolarity and hydroperoxides on pHves. The data suggest that hepatocellular hydration affects the proton gradients built up across the membranes of endocytotic FITC-dextran-accessible compartments in a microtubule-dependent way. They further suggest that hydroperoxides induce vesicular acidification in a colchicine- and Ba(2+)-sensitive way. Because hydroperoxides induce Ba(2+)-sensitive cell shrinkage [Hallbrucker, Ritter, Lang, Gerok and Häussinger (1992) Eur. J. Biochem. 211, 449-458], the results are compatible with the view that hydroperoxide-induced cell shrinkage mediates vesicular acidification. It is concluded that modulation of vesicular pH by the hepatocellular hydration state may play a role in triggering some metabolic changes in response to cell swelling/shrinkage.
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Xu, K., R. M. Williams, D. Holowka, and B. Baird. "Stimulated release of fluorescently labeled IgE fragments that efficiently accumulate in secretory granules after endocytosis in RBL-2H3 mast cells." Journal of Cell Science 111, no. 16 (1998): 2385–96. http://dx.doi.org/10.1242/jcs.111.16.2385.
Full textAbstract:
Sensitization of RBL-2H3 mast cells with monomeric fluorescein-5-isothiocyanate (FITC)-labeled immunoglobulin E (IgE) results in slow but highly efficient accumulation of labeled IgE fragments in a pool of acidic peripheral vesicles that are visible by fluorescence microscopy after raising endosomal pH with ammonium chloride. Stimulation of cells containing these FITC-IgE fragments by aggregation of high affinity receptors for IgE (FcepsilonRI) or by Ca2+ ionophore and phorbol 12-myristate 13-acetate results in release of FITC fluorescence from the cells, which can be monitored continuously with a spectrofluorometer. The fluorescence release process corresponds to cellular degranulation: it is prevented under conditions that prevent stimulated beta-hexosaminidase release, and these two processes exhibit the same antigen dose-dependence and kinetics. Pulse-chase labeling reveals that aggregation of FITC-IgE bound to FcepsilonRI at the cell surface causes internalization and delivery to the regulated secretory vesicles with a high efficiency similar to monomeric IgE-FcepsilonRI, but more rapidly. Binding of Cy3-modified IgE to FcepsilonRI results in labeling of the same secretory vesicles as in FITC-IgE-sensitized cells, and these Cy3-labeled vesicles can be observed by fluorescence microscopy without neutralization of intracellular compartments. Simultaneous three-photon microscopy of serotonin fluorescence and two-photon microscopy of Cy3 fluorescence reveals that these Cy3-labeled vesicles coincide with serotonin-labeled secretory granules. After stimulation of the cells via aggregation of IgE-FcepsilonRI or addition of Ca2+ ionophore and phorbol 12-myristate 13-acetate, depletion of the Cy3 label from the intracellular vesicles is observed with confocal microscopy. These results provide strong evidence for the lysosomal nature of secretory granules in these cells. In addition, they provide the basis for a direct, real-time method for monitoring single cell degranulation.
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Sun, YY. "Effects of Combined or Along VFA, pH, Lipopolysaccharide and Histamine on the Rumen Epithelial Permeability of Dairy Goats In Vitro." International Journal of Agriculture and Biology 26, no. 01 (2021): 79–86. http://dx.doi.org/10.17957/ijab/15.1811.
Full textAbstract:
This study investigated whether concurrent presence of lipopolysaccharide (LPS) and histamine (HIS) have the potential to increase permeability of the ruminal epithelium at physiological pH and acidotic ruminal pH. Nine 2.5-year-old female lactating Saanen dairy goats (42.79 ± 5.61 kg of BW; Mean ± SD) were used as a ruminant model. ruminal epithelium of goats were collected and mounted in Ussing chambers on their mucosal side in different gradient buffer solutions (pH 7.4, 5.5 and 5.2) containing LPS (0, 30 and 60 KEU·mL-1) or HIS (0, 0.5 and 10 ng·mL-1). The rumen epithelial electrophysiological indexes, such as short-circuit (Isc), tissue conductance (Gt) and the permeability of marker molecules of different sizes (horseradish peroxidase, HRP and fluorescein 5(6)-isothiocyanate, FITC) from the mucosal to the serosal side, were measured. Both Isc and Gt were increased, accompanied by enhanced flux of FITC, with a decrease of mucosal pH (P < 0.05). The addition of LPS at mucosal pH 5.2 significantly increased Isc, Gt and FITC flux rates and decreased potential difference (PD) (P < 0.05). Additionally, the concurrent presence of LPS and HIS at both physiological and acidotic ruminal pH also significantly increased the permeability of ruminal epithelium asevidenced by increasing Isc, Gt and FITC flux rates and decreasing PD. In summary, our results have shown that concurrent presence of LPS 60 KEU‧mL-1 and HIS 10 ng‧mL-1 at mucosal pH 5.5 can increase the permeability of ruminal epithelium. The combination of low pH and both high LPS and HIS may increase vulnerability to aggravated rumen epithelial barrier dysfunction. © 2021 Friends Science Publishers
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Wu, Jung-He, and Scott L. Diamond. "Fluorogenic Fibrinogen and Fibrin Facilitate Macromolecular Assembly and Dynamic Assay of Picomolar Levels of Plasminogen Activators under Well Mixed Conditions." Thrombosis and Haemostasis 74, no. 02 (1995): 711–17. http://dx.doi.org/10.1055/s-0038-1649802.
Full textAbstract:
SummaryFibrinogen labeled with fluorescein isothiocyanate (FITC) was tested for its ability to serve as a template for macromolecular assembly as well as to provide a fluorogenic signal to allow continuous monitoring of plasminogen activation and fibrinolysis. As dilute solutions of FITC-fibrinogen or FITC-fibrin fiber suspension were degraded during lysis, release of fluorescent fragments abolished proximity- based quenching and resulted in a 2.0- or 3.6-fold increase in fluorescence intensity, respectively. Addition of plasmin at a final concentration of 10 pM to FITC-fibrinogen (10 nM) produced a detectable level of fluorescence dequenching. The assay had sufficient sensitivity to detect plasmin activity in the presence of excess antiplasmin activity, indicating the dissociation of a reversible antiplasmin-plasmin complex. The detection limit of the reaction assay was 20 pM and 200 pM of recombinant tPA and urokinase, using 10 nM FITC-fibrin and 10 nM and 5 nM plasminogen, respectively. The 10-fold greater sensitivity of the assay for tPA was likely due to the molecular assembly of tPA and plasminogen on the FITC-fibrin. Addition of thrombin (1 U/ml) and plasmin (0.1 nM) to 10 nM FITC-fibrinogen produced fluorescence quenching at first due to fibrinogen polymerization followed by dequenching due to fibrinolysis. Addition of 10 mM ∈-aminocaproic acid to mixtures of thrombin and plasmin allowed the quenching assay of thrombin activity in the presence of active plasmin. FITC-fibrinogen could be copolymerized with recalcified platelet poor plasma (isolated from citrated whole blood) to yield fibrin that was fluorogenic. Dequenching was observed when plasmin was used to degrade the fibrin formed from the platelet poor plasma. Given the large signal generated upon degradation of the fluorogenic fibrin(ogen), at least 105 determinations can be run from 100 mg of FITC-labeled fibrinogen using a standard fluorimeter and 0.1 to 3.0 ml reaction volumes. The versatility of the fluorogenic fibrinogen substrate allowed the configuration of assays to detect and measure the activity of thrombin, plasmin, tPA, uPA, and α2-antiplasmin. The ability to assemble blood proteins on a fluorogenic fibrinogen or fibrin template provides unique opportunities for the dynamic study of binding and enzymatic events on the fibrin surface under well mixed conditions.
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González-García, Inés, Beatriz Rodríguez-Bayona, Francisco Mora-López, Antonio Campos-Caro, and José A. Brieva. "Increased survival is a selective feature of human circulating antigen-induced plasma cells synthesizing high-affinity antibodies." Blood 111, no. 2 (2008): 741–49. http://dx.doi.org/10.1182/blood-2007-08-108118.
Full textAbstract:
The present study shows that tetanus toxoid (tet) booster releases to the human circulation 2 subsets of specific plasma cells (PCs), as defined by phenotype and morphology, which clearly differed in the staining capacity of their cytoplasmic antibodies (Abs) with fluorescein isothiocyanate (FITC)–labeled tet–fragment C (tetC). These cells, called tetCHIGH and tetCINT PCs according to their either high or intermediate FITC-tetC staining capacity, exhibit similar rapid temporary kinetics in the blood (5-8 days after boost), contain many cycling cells, express equivalent amounts of BLIMP-1 mRNA, and produce similar quantities of IgG. However, Abs synthesized by tetCHIGH PCs show a tetC affinity more than 10 times higher than that exhibited by tetCINT PC Abs, and indicated by IGVH sequence analysis. Chemotaxis to CXCL12, a requisite for bone marrow (BM) PC homing, is similar for both cell types. Circulating nonspecific and tetCINT PCs, but not tetCHIGH PCs, tend to undergo spontaneous apoptosis, as demonstrated by APO2.7 and activated caspase-3 expression, and cell recovery. These results indicate that tet booster generates 2 discrete subsets of specific PCs exhibiting different ranges of Ab affinity for the immunogen, and that only those synthesizing high-affinity Abs show enhanced survival. This inherent property may be essential for determining the BM fate of PCs secreting high-affinity Ab.
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Wang, Junyao, Lu-lu Han, Ye-ming Sun, and Tian-yi Su. "Alignment System and Application for a Micro/Nanofluidic Chip." Micromachines 9, no. 12 (2018): 621. http://dx.doi.org/10.3390/mi9120621.
Full textAbstract:
In this paper, a direct pre-bonding technology after alignment of the chip is presented to avoid the post-misalignment problem caused by the transferring process from an alignment platform to a heating oven. An alignment system with a high integration level including a microscope device, a vacuum device, and an alignment device is investigated. To align the chip, a method of ‘fixing a chip with microchannels and moving a chip with nanochannels’ is adopted based on the alignment system. With the alignment system and the assembly method, the micro/nanofluidic chip was manufactured with little time and low cost. Furthermore, to verify the performance of the chip and then confirm the practicability of the device, an ion enrichment experiment is carried out. The results demonstrate that the concentration of fluorescein isothiocyanate (FITC) reaches an enrichment value of around 5 μM and the highest enrichment factor is about 500-fold. Compared with other devices, an alignment system presented in this paper has the advantages of direct pre-bonding and high integration level.
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Kim, D., and W. N. Duran. "Platelet-activating factor stimulates protein tyrosine kinase in hamster cheek pouch microcirculation." American Journal of Physiology-Heart and Circulatory Physiology 268, no. 1 (1995): H399—H403. http://dx.doi.org/10.1152/ajpheart.1995.268.1.h399.
Full textAbstract:
We studied the interactions between platelet-activating factor (PAF) and protein tyrosine kinase (PTK) in the modulation of microvascular responses in the hamster cheek pouch using intravital microscopy and computer-assisted image analysis. Changes in arteriolar diameter and in integrated optical intensity (IOI; an index of vascular permeability) were measured. Fluorescein isothiocyanate-labeled dextran 150 (FITC-Dx 150) served as a tracer for macromolecular transport. Genistein and tyrphostin 25, two PTK inhibitors, were applied topically in separate experiments. Pretreatment with 10(-4), 10(-6), and 10(-8) M genistein and with tyrphostin 25 at 10(-5) and 10(-7) M attenuated the maximal increment in mean IOI (+/- SE) induced by PAF at 10(-7) M (19.9 +/- 5.3, 21.5 +/- 4.5, 58.5 +/- 11.4, 28.7 +/- 7.6, and 35.0 +/- 10.9 vs. 70.7 +/- 8.9 units, respectively). Pretreatment with PTK inhibitors resulted in vasodilation but did not inhibit PAF-induced vasoconstriction. Our results suggest that PTK represents a biochemical pathway involved in the PAF modulation of microvascular permeability but not PAF modulation of arteriolar tone.
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Shoji, Y., J. Shimada, Y. Mizushima, et al. "Cellular uptake and biological effects of antisense oligodeoxynucleotide analogs targeted to herpes simplex virus." Antimicrobial Agents and Chemotherapy 40, no. 7 (1996): 1670–75. http://dx.doi.org/10.1128/aac.40.7.1670.
Full textAbstract:
In this study, we synthesized antisense oligodeoxynucleotides (ODNs) with phosphodiester, phosphorothioate (S-ODNs), or methylphosphonate linkages complementary to the splicing acceptor site of immediate-early pre-mRNA 5 of herpes simplex virus type 1 (HSV-1). The antiviral activity of each analog on cytopathic effect in cells infected with HSV-1 or HSV-2 was assessed and compared with the cellular uptake of the analog. We found that antisense S-ODNs showed the most potent antiherpetic activity, with 50% inhibitory concentrations of 5 microM for HSV-1 and 0.25 microM for HSV-2. The antiviral effect of antisense S-ODNs was stronger and longer acting than that of acyclovir. Cell association of S-ODNs was the highest and paralleled antiviral activity. Furthermore, some fluorescein isothiocyanate (FITC)-labeled S-ODNs were recognized in the nuclei in HSV-1 infected cells by confocal laser scanning microscopy. S-ODNs located in the nucleus could access the targeted mRNA, which might be responsible for the antiviral activities. Although our study also showed non-sequence-specific activity, which implies that multiple mechanisms are involved, S-ODNs are a promising novel anti-herpetic agent.
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Tomokiyo, Kazuhiko, Yuichi Kamikubo, Takako Hanada, et al. "Von Willebrand factor accelerates platelet adhesion and thrombus formation on a collagen surface in platelet-reduced blood under flow conditions." Blood 105, no. 3 (2005): 1078–84. http://dx.doi.org/10.1182/blood-2004-05-1827.
Full textAbstract:
AbstractPlasma von Willebrand factor (VWF) has been identified as an indispensable factor for platelet adhesion and thrombus formation on a collagen surface under flow conditions. VWF binds to collagen and then tethers platelets to the collagen surface through interaction with platelet glycoprotein Ib and also contributes to the thrombus formation on the collagen surface. In the present study, we demonstrated that the addition of VWF/factor VIII complex or purified VWF (&gt; 2 ristocetin cofactor activity units/mL) increased platelet adhesion to the collagen surface in platelet-reduced blood (∼ 5 × 104 platelets/μL) to the normal level. VWF had no stimulatory effect when it was allowed to bind to the collagen surface before blood flow was initiated. Addition of an excess of FITC (fluorescein-5-isothiocyanate)–abeled VWF to platelet-reduced blood under these flow conditions demonstrated that the VWF was mainly incorporated into the platelet aggregates. These results indicated that the supplemented VWF stimulates the platelet adhesion onto the collagen surface by enhancing platelet aggregation in the platelet-reduced condition. This also suggests a possibility that supplementation of VWF to individuals with thrombocytopenia might be effective for increasing their hemostatic potential.
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Zhuang, Hua, Jing Wu, Wei Xu, et al. "Transfection of CTGF siRNA inhibits transdifferentiation in human lens epithelium cell line B3 in vitro." International Eye Research 1, no. 1 (2020): 17–23. http://dx.doi.org/10.18240/ier.2020.01.04.
Full textAbstract:
AIM: To investigate the expression of connective tissue growth factor (CTGF) and α-smooth muscle actin (α-SMA) in human lens epithelium cell (HLEC) line B3 after transfection by liposome-coated small interfering RNA (siRNA) targeting CTGF. METHODS: HLECs were transfected with siRNA targeting CTGF, labeled with 5’-fluorescein isothiocyanate (5’-FITC) and coated with lipofectamine. The transfection ratio was evaluated via fluorescence intensity. Cell counting kit-8 (CCK-8) assay was performed to assess cytoviability of both non-transfected and transfected HLECs. Quantitative reverse transcription-polymerase chain reaction (RT-PCR), cell immunochemistry and Western blot analysis were conducted to detect the expression changes of CTGF and α-SMA after transfection. RESULTS: A highly effective transfection ratio was observed in siRNA co-transfected with lipofectamine. The transfection ratio reached 95% at 24h. The proliferation of HLECs was inhibited by siRNA after 72h transfection. The expression of CTGF and α-SMA significantly decreased in HLECs after transfected by CTGF siRNA for 24h. This effect was not found in negative control siRNA. CONCLUSION: siRNA targeting CTGF decrease CTGF and α-SMA expression in HLECs, which is a potential therapeutic strategy for posterior capsular opacification.
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Hempel, A., T. Noll, A. Muhs, and H. M. Piper. "Functional antagonism between cAMP and cGMP on permeability of coronary endothelial monolayers." American Journal of Physiology-Heart and Circulatory Physiology 270, no. 4 (1996): H1264—H1271. http://dx.doi.org/10.1152/ajpheart.1996.270.4.h1264.
Full textAbstract:
The role of the intracellular second messengers guanosine 3', 5'-cyclic monophosphate (cGMP) and adenosine 3', 5'-cyclic monophosphate (cAMP) in the control of macromolecule permeability was studied in cultured monolayers of microvascular coronary endothelial cells from rat. Macromolecule permeability was determined as passage of fluorescein isothiocyanate (FITC)-labeled albumin across the monolayers. Activation of adenylyl cyclase by the beta-adrenoceptor agonist isoproterenol (Iso; 10(-5) M) and the A2-adenosine receptor agonist 5'-(N-ethylcarboxamido)-adenosine (NECA; 10(-7) M) induced an increase in cellular cAMP contents that was accompanied by an increase in albumin flux. Effects of Iso and NECA on cellular cAMP level and albumin flux could be antagonized by a stimulator of the particular guanylyl cyclase, atrial natriuretic peptide (ANP; 10(-7) M), and stimulators of the soluble guanylyl cyclase, 3-morpholinosydnonimine (SIN-1; 10(-7) M) and sodium nitroprusside (SNP; 10(-6) M). ANP, SIN-1, and SNP also reduced cAMP content and basal macromolecule flux in unstimulated monolayers. 8-Bromoguanosine 3', 5'-cyclic monophosphate (8-BrcGMP; 5 x 10(-6) M), a stimulator of protein kinase G, reduced the increase in albumin flux under Iso (10(-5) M), NECA (10(-7) M), or 8-bromoadenosine 3', 5'-cyclic monophosphate (8-BrcAMP; 5 x 10(-6) M). The present study shows that cGMP and cAMP are functional antagonists in the control of macro molecule permeability.
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Liang, Ying, Xiang-Wei Fu, Jun-Jie Li, Dian-Shuai Yuan, and Shi-En Zhu. "DNA methylation pattern in mouse oocytes and their in vitro fertilized early embryos: effect of oocyte vitrification." Zygote 22, no. 2 (2012): 138–45. http://dx.doi.org/10.1017/s0967199412000512.
Full textAbstract:
SummaryThis study was conducted to investigate the pattern of DNA methylation in vitrified–thawed mouse oocytes and their in vitro fertilized early embryos. Firstly, mouse oocytes at metaphase II (MII) stage of meiosis were allocated randomly into three groups: (1) untreated (control); (2) exposed to vitrification solution without being plunged into liquid nitrogen (toxicity); or (3) vitrified by open-pulled straw (OPS) method (vitrification). Oocytes from all three groups were fertilized subsequently in vitro. The level of DNA methylation in the MII oocytes and their early embryos was then examined by immunofluorescence using an anti-5-methylcytosine (anti-5-MeC) monoclonal antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Developmental rates to 2-cell embryos (62.28%) and blastocysts (43.68%) of the vitrified–thawed oocytes were lower (P < 0.01) than those of fresh oocytes (81.47%, 61.99%) and vitrification solution treated (79.20%, 60.04%) oocytes. DNA methylation (as reflected by 5-MeC fluorescence intensity) in the vitrification group was less (P < 0.01) for MII oocyte and 2- to 8-cell stages compared with that in the control and toxicity groups. Accordingly, a reduction in global genomic methylation due to vitrification of MII oocytes may result in compromised in vitro developmental potential in early mouse embryos.
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Oliveira, Raylson Pereira de, Nínivy Marques Soares, José Givanildo da Silva, et al. "Occurrence of anti-Neospora caninum antibodies in cattle in the dairy farming region of the state of Piauí, Brazil." Revista Brasileira de Parasitologia Veterinária 27, no. 4 (2018): 589–92. http://dx.doi.org/10.1590/s1984-296120180046.
Full textAbstract:
Abstract This study focused on the detection of anti-Neospora caninum IgG antibodies in cows in the dairy farming region of the state of Piauí, Brazil. To this end, serum samples were collected from 255 dairy cows on 17 farms located in the dairy farming region of the municipality of Parnaíba, Piauí, Brazil. The indirect fluorescence antibody test (IFAT) was employed to detect anti-N. caninum IgG antibodies, using anti-bovine IgG (Sigma®) conjugated to fluorescein isothiocyanate (FITC) and a cutoff point of 1:200. Of the 255 samples analyzed, 69 (27.06%) were positive for anti- N. caninum IgG antibodies, the relative frequency found by property was: 1 (20.00%), 2 (53.33%), 3 (46.66%), 4 (53.33%), 5 (26.66%), 6 (6.66%), 7 (6.66%), 8 (20.00%), 9 (26.66%), 10 (26.66%), 11 (20.00%), 12 (20.00%), 13 (46.66%), 14 (26.66%), 15 (26.66%), 16 (20.00%) and 17 (13.33%). with titers of 200 (15.94%), 400 (20.30%), 800 (24.63%), 1600 (23.18%) and 3200 (15.94%), being the highest frequency for the titer of 800. This study demonstrates for the first time that cows from dairy herds of Parnaíba municipality, state of Piauí, are exposed to N. caninum.
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Murphy, Timothy V., Brian E. Spurrell, and Michael A. Hill. "Tyrosine phosphorylation following alterations in arteriolar intraluminal pressure and wall tension." American Journal of Physiology-Heart and Circulatory Physiology 281, no. 3 (2001): H1047—H1056. http://dx.doi.org/10.1152/ajpheart.2001.281.3.h1047.
Full textAbstract:
Arterioles respond to increased transmural pressure with myogenic constriction. The present study investigated the role of tyrosine phosphorylation in myogenic activity. Cannulated segments of a rat cremaster arteriole were fixed under pressure, followed by incubation with fluorescein isothiocyanate (FITC)-conjugated anti-phosphotyrosine. Smooth muscle cell fluorescence intensity was measured with the use of confocal laser-scanning microscopy. Anti-phosphotyrosine fluorescence intensity in muscle cells of arterioles maintained at 100 mmHg was reduced by the tyrosine kinase inhibitor tyrphostin A47 (30 μM) and increased by the tyrosine phosphatase inhibitor pervanadate (100 μM). In time-course experiments, anti-phosphotyrosine fluorescence increased slowly (over 5 min) after an acute increase in intraluminal pressure, and was dissociated from myogenic contraction (within 1 min). In contrast, angiotensin II (0.1 μM) caused rapid constriction and increased tyrosine phosphorylation. Anti-phosphotyrosine fluorescence was also pressure dependent (10–100 mmHg). Abolition of myogenic activity, either through removal of extracellular Ca2+, or exposure to verapamil (5 μM) or forskolin (0.1 μM) caused a further increase in anti-phosphotyrosine fluorescence. We conclude that transmural pressure and/or wall tension in arterioles causes increased tyrosine phosphorylation; however, this is not involved in the acute phase of myogenic constriction but may be involved in later responses, such as sustained myogenic tone or mechanisms possibly related to growth.