Chemical and Physical Variables Affecting Fluorescein Isothiocyanate and Its Protein Conjugates

Klugerman, Maxwell R.

doi:10.21236/ad0459385

Cited by 14 publications

(6 citation statements)

References 2 publications

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“…The relative fluorescence of F-Gal was 0.3 times that of free FITC. The yield is consistent with the observations that fluorescence of fluorescein and most other dyes is typically decreased by >50% upon conjugation (33). The functional properties of F-Gal were analyzed in (1) radioligand competition assays with intact CHO cells expressing rGalR1 and with membrane preparations from COS-7 cells transiently transfected with rGalR1 cDNA; (2) functional assays for inhibition of forskolin-stimulated cAMP production in rGalR1/CHO cells; and 3) saturation binding of F-Gal to rGalR1/CHO cells measured with flow cytometry.…”

Section: Resultssupporting

confidence: 89%

Evidence for Hydrophobic Interaction between Galanin and the GalR1 Galanin Receptor and GalR1-Mediated Ligand Internalization: Fluorescent Probing with a Fluorescein−Galanin

Wang¹,

Clemmons²,

Strader³

et al. 1998

Biochemistry

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Galanin is a neuropeptide that activates specific receptors to modulate several physiological functions including food intake, nociception, and learning and memory. The molecular nature of the interaction between galanin and its receptors and the fate of the galanin/receptor complex after the binding event are not understood. A fluorescein-N-galanin (F-Gal) was generated to measure the interaction between galanin and rat GalR1 galanin receptor (rGalR1) and rGalR1-mediated ligand internalization using flow cytometry in transfected Chinese hamster ovary (CHO) cells. Like galanin, F-Gal bound rGalR1 with high affinity and stimulated intracellular signaling events. Fluorescence quenching by soluble KI of rGalR1-bound F-Gal revealed a highly protected environment around the fluorescein, suggesting that the N-terminal portion of galanin, which constitutes the binding site of galanin for the receptor, binds to a protected hydrophobic binding pocket within the receptor. Exposure to F-Gal stimulated rapid (t1/2 approximately 10 min) and extensive (78%) internalization of surface F-Gal into rGalR1/CHO cells at 37 degreesC but not at 0 degreesC. In addition, the internalization did not occur in parental CHO cells at either 0 or 37 degreesC and was inhibited by addition of 0.25 M sucrose in the medium, indicating a GalR1-mediated energy-requiring endocytic process. These results revealed a hydrophobic interaction between galanin and the GalR1 receptor, which is in contrast to those of other G protein-coupled receptors that mainly require hydrophilic interaction with their peptide ligands near or outside the plasma membrane surface, and illustrated that the initial binding interaction is followed by rapid cellular internalization of the agonist/GalR1 complex.

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Section: Resultssupporting

confidence: 89%

Evidence for Hydrophobic Interaction between Galanin and the GalR1 Galanin Receptor and GalR1-Mediated Ligand Internalization: Fluorescent Probing with a Fluorescein−Galanin

Wang¹,

Clemmons²,

Strader³

et al. 1998

Biochemistry

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“…Thin-layer chromatographic separation of the low-molecular-weight-labeled fraction (see Materials and Methods) showed that the major amine-containing phospholipids of sea urchin sperm (21), phosphatidyl ethanolamine and phosphatidyl serine, which are both potential targets for ~25IFC-labeling, were not labeled when compared with authentic standards of IFC-labeled phosphatidyl ethanolamine (Rf = 0.48) and IFC-labeled phosphatidyl serine (Rf = 0.26) (data not shown). By similar chromatography we found that <10% of the total radioactivity present in the NP-40 extract was free ~25IFC, and none of it was ~25I diiodoflu-orescein amine (Rf = 0.4) (data not shown), the expected hydrolysis product of t25IFC (20,26). Thus little ~2~IFC was noncovalently bound to sperm, and the derivatized species were stable throughout the fractionation procedure.…”

Section: Fractionation Of T2slfc-labeled Sperm Componentsmentioning

confidence: 75%

“…It is thus worth considering some potential artifacts that could result from the t25IFC-labeling protocol we employed. Artifactual loss of ~25IFC-labeled proteins recoverable with the anti-IFC immunoadsorbent could be the result of degradation of the thiourea derivative (i.e., that formed by the reaction of isothiocyanates with amino groups); however, the extremes in pH and temperature necessary to degrade these derivatives (20,26) are not likely to be encountered in embryos. Aryl halides, like t2~IFC, are generally very stable (27); nonetheless, the ovoperoxidase released at fertilization (9) could provide an enzymatic means of deiodinating the ~25IFC.…”

Section: Discussionmentioning

confidence: 99%

Sperm surface proteins persist after fertilization.

Gundersen¹,

Shapiro²

1984

The Journal of Cell Biology

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Certain sperm components labeled with fluorescein isothiocyanate or its radioactive derivative, 1251-diiodofluorescein isothiocyanate (t251FC), are transferred at fertilization to the egg, where they persist throughout early cleavage stages at a localized site in the embryo cytoplasm (Gabel, C. A., E. M. Eddy, and B. M. Shapiro, 1979, Cell, 18:207-215; Gundersen, G. G., C. A. Gabel, and B. M. Shapiro, 1982, Dev. Biol., 93:59-72). By using image intensification we have extended these observations in the sea urchin to the pluteus larval stage, in which >60% of the embryos have localized fluorescent sperm components. Because of the unusual persistence of the sperm components in the embryo, a characterization of the nature of the labeled species in sea urchin sperm was undertaken. ~10% of the 1251FC was in sperm polypetides of Mr > 15,000. These proteins were on the sperm surface as shown by their sensitivity to externally added proteases. The remainder of the ~251FC in sperm was in several low-molecular-weight species, none of which was ~251FC-derivatized phospholipid.To determine if any labeled sperm polypeptides remained intact in the embryo after fertilization, ~251FC-labeled sperm proteins were recovered from one-cell and late gastrula stage embryos by using an anti-IFC immunoadsorbent. Most of the labeled sperm proteins were degraded shortly after fertilization; however, distinct sets of labeled polypeptides were recovered from both one-cell and gastrula stage embryos. Six of the labeled polypeptides recovered from both embryonic stages had identical SDS gel mobilities as labeled sperm polypeptides. Other polypeptides in the embryos appeared to arise from limited proteolysis of sperm proteins. Thus, in this physiological cell fusion system, individual sperm proteins are transferred to the egg at fertilization, and some persist intact or after specific, limited degradation long after gamete fusion, until at least the late gastrula stage.Fertilization is the process whereby haploid genomes are united and development is initiated. Central to this process is the fusion of gamete membranes, which results in the incorporation of the sperm nucleus as well as other, nonnuclear, sperm components into the egg. Although the insertion of cytoplasmic sperm components during fertilization has been observed in almost every animal phyla, few examples exist in which the fates of these components have been addressed (see bibliographies in references l, 33, 34). Furthermore, the great dilution of sperm components after gamete fusion (from 104-to 10z1-fold depending on the species, [1]) has limited such studies to observations of morphological markers of the sperm, such as the axoneme or the mitochondfia, which can be identified after fertilization. The consensus of these investigations is that sperm organelles are degraded during the early cleavage stages, although notable exceptions do exist (see bibliographies in references 33 and 34). In none of these studies have individual sperm components been pursued at the mole...

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“…To monitor tissue explant permeability, we used DCF, a slightly polar, small molecular weight (400 kD) compound that has no known mammalian transporter. The measured equilibrium concentration of DCF was below that which was expected by mass balance, and we believe that the incomplete recovery was due to DCF binding membrane and culture medium proteins [38] as well plastic culture-ware. DCF may cross the monolayer through altered intercellular tight junctions or any number of transcellular channels.…”

Section: Discussionmentioning

confidence: 99%

Pre-B cell colony enhancing factor (PBEF/NAMPT/Visfatin) and vascular endothelial growth factor (VEGF) cooperate to increase the permeability of?the?human placental amnion

2013

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Fluid efflux across the region of the amnion overlying the placenta is an essential component of the intramembranous absorption pathway that maintains amniotic fluid volume homeostasis. Dysregulation of this pathway may result in adverse pregnancy outcomes, however the factors controlling amnion permeability are unknown. Here, we report a novel mechanism that increases placental amnion permeability. Pre-B Cell Colony Enhancing Factor (PBEF) is a stress-responsive cytokine expressed by the human amnion, and is known to induce Vascular Endothelial Growth Factor (VEGF) production by other cell types. Interestingly, VEGF is up-regulated in the ovine amnion when intramembranous absorption is augmented. In this study, we show that PBEF induced VEGF secretion by primary human amniotic epithelial cells (AEC) derived from the placental amnion, as well as from the reflected amnion that lines the remainder of the gestational sac. Further, PBEF treatment led to the increased expression of VEGFR2 in placental AEC, but not reflected AEC. To test the hypothesis that PBEF and VEGF increase placental amnion permeability, we monitored the transfer of 2′,7′-dichlorofluorescein (DCF) from the fetal to the maternal side of human amnion explants. A treatment regimen including both PBEF and VEGF increased the rate of DCF transfer across the placental amnion, but not the reflected amnion. In summary, our results suggest that by augmenting VEGFR2 expression in the placental amnion, PBEF primes the tissue for a VEGF-mediated increase in permeability. This mechanism may have important implications in amniotic fluid volume control throughout gestation.

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Chemical and Physical Variables Affecting Fluorescein Isothiocyanate and Its Protein Conjugates

Cited by 14 publications

References 2 publications

Evidence for Hydrophobic Interaction between Galanin and the GalR1 Galanin Receptor and GalR1-Mediated Ligand Internalization: Fluorescent Probing with a Fluorescein−Galanin

Evidence for Hydrophobic Interaction between Galanin and the GalR1 Galanin Receptor and GalR1-Mediated Ligand Internalization: Fluorescent Probing with a Fluorescein−Galanin

Sperm surface proteins persist after fertilization.

Pre-B cell colony enhancing factor (PBEF/NAMPT/Visfatin) and vascular endothelial growth factor (VEGF) cooperate to increase the permeability of?the?human placental amnion

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