Fluorescence of the natural dye saffron: Selective reaction with eosinophil leucocyte granules

Treatment of methanol-fixed chicken, rat, horse and human blood smears with saturated solutions of saffron in borate buffer at pH 10 results in a bright yellow-green fluorescence reaction of the acidophilic cytoplasm granules in mammalian eosinophils and chicken heterophils under violet-blue exciting light. Spectral characteristics of saffron (emission peak at 543 nm under 436 nm excitation) and its selective fluorescence with acidophilic structures support the possibility of employing this old microscopic stain as a new fluorochrome.     

Fluorescence of mast cell granules in paraffin sections and cell smears induced by an N-quaternary oxazole scintillator

Summary The N-quaternized derivative of dimethyl-POPOP (termed Q4) induces a bluish-green fluorescent reaction in mast cell granules from paraffin sections and cell smears, in addition to a previously described bluish-white fluorescent reaction in chromatin DNA. The chromatin reaction was abolished by staining the samples either with Mayer's Haematoxylin before Q4 treatment or by Q4 treatment at pH 1.5. The reaction in mast cell granules was absent after substrate methylation. The staining sequence Haematoxylin-Eosin-Q4 also worked well in paraffin sections, allowing the observation of the current histological image under bright-field illumination as well as double-colour emission under fluorescence microscopy. The sequence is proposed as a new diagnostic procedure for demonstrating mast cell granules.     

Growth inhibition of mammalian cells by eosinophil cationic protein.

Eosinophil cationic protein (ECP), one of the major components of basic granules of eosinophils, is cytotoxic to tracheal epithelium. However, the extent of this effect on other cell types has not been evaluated in vitro. In this study, we evaluated the effect of ECP on 13 mammalian cell lines. ECP inhibited the growth of several cell lines including those derived from carcinoma and leukemia in a dose-dependent manner. The IC(50) values on A431 cells, MDA-MB-453 cells, HL-60 cells and K562 cells were estimated to be approximately 1-5 microm. ECP significantly suppressed the size of colonies of A431 cells, and decreased K562 cells in G1/G0 phase. However, there was little evidence that ECP killed cells in either cell line. These effects of ECP were not enhanced by extending its N-terminus. Rhodamine B isothiocyanate-labeled ECP started to bind to A431 cells after 0.5 h and accumulated for up to 24 h, indicating that specific affinity for the cell surface may be important. The affinity of ECP for heparin was assessed and found to be reduced when tryptophan residues, one of which is located at a position in the catalytic subsite of ribonuclease in ECP, were modified. The growth-inhibitory effect was also attenuated by this modification. These results suggest that growth inhibition by ECP is dependent on cell type and is cytostatic.     

Fluorescence energy transfer between subfragment-1 and actin points in the rigor complex of actosubfragment-1.

The fast-reacting thiol (SH1) of myosin subfragment-1 (S-1) was covalently and specifically labeled with (iodoacetamido)fluorescein (IAF), while Cys-373 of actin was also covalently and preferentially labeled with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (1,5-IAEDANS). The method of fluorescence energy transfer was used to examine the spatial proximity between the two sites, i.e., SH1 and Cys-373, in the rigor complex of acto-S-1. Approximately 30% fluorescence energy transfer was observed from the 1,5-IAEDANS on actin as a donor to the IAF on S-1 as an acceptor in their rigor complex; under certain assumptions this corresponds to a distance of ca. 6.0 nm.     

An in vitro reversible interconversion of rat liver fatty acid binding protein having different isoelectric points by virtue of the fatty acid content.

Rat liver fatty acid binding protein (FABP) was purified to homogeneity by procedures including Sephadex G-100 and DEAE-cellulose column chromatographies. FABP was resolved into two major peaks, A and B, by the first DEAE-cellulose column chromatography. Each of these two fractions exhibited apparent homogeneity upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate with a molecular weight of 14,000 Da and amino acid analysis of these fractions has revealed that they are virtually identical or closely resemble each other. However, their fatty acid content was significantly different and heterogeneity was clearly demonstrated in the patterns of isoelectric focusing. In this communication, a single isoform (pI 5.0) from peak B FABP was further purified by successive DEAE-cellulose column chromatography and used as the final preparation. When the final FABP was partly freed of fatty acids by a mild delipidation technique using Lipidex 1,000, the pI shifted upward from 5.0 to 7.0. However, the pI of the delipidated FABP returned to its original pI of 5.0 after recombining fatty acids. These in vitro manipulations of bound fatty acid content made clear its possible cause of the microheterogeneity of FABP.     

Selective regulation of eosinophil degranulation by interleukin 1 beta.

Recent evidence confirms that cytokines such as IL-1, IL-4, IL-5, and GM-CSF may enhance or inhibit eosinophil function. Functions that are susceptible to modulation include eosinophil-mediated antibody-dependent damage of helminthic parasites, oxidative metabolism and degranulation. We have employed IgG and IgE-coated Sepharose beads to investigate selective modulation of IgG and IgE-mediated enzyme release by IL-1 beta. Both IgG and IgE-coated beads induced release of granular enzymes beta-glucuronidase and arylsulfatase. Enzyme release from IgG-stimulated eosinophils was inhibited by preincubation with IL-1 beta (100 pg/ml, P less than or equal to 0.05). In contrast, enzyme release by IgE-stimulated eosinophils was enhanced by IL-1 beta (100 pg/ml, P less than or equal to 0.05). These studies support the hypothesis that IL-1 beta has specific selective actions on eosinophil function. Furthermore, these actions on particle-stimulated enzyme release suggest that IgG and IgE mediated processes in eosinophils are differentially regulated.     

Precipitation of egg white proteins below their isoelectric points by sodium dodecyl sulphate and temperature.

The precipitating of effect of sodium dodecyl sulphate (SDS) on the egg white proteins ovalbumin, conalbumin and lysozyme was studied at 25 degrees C and at different pH values. The proteins precipitated below their respective isolectric points, provided the detergent to protein ratio was appropriate. The pH profile of precipitation was different for the three proteins reflecting net charge differences. The binding of SDS to the proteins was studied with [35S]-labelled SDS and for ovalbumin a ratio of 21--28 SDS molecules/protein molecule, dependent on the concentration of SDS initially used, seem to be required for precipitation at pH 4.5. This number, however, is dependent on pH and increases with an increased positive net charge of the protein. The precipitating effect of SDS was identical for ovalbumin, conalbumin and lysozyme when compared on a gram to gram basis (0.1--0.15 g SDS/g precipitated protein). The precipitated protein was denatured as measured by differential scanning calorimetry, but was also completely redissolved if pH was increased to above the isoelectric point. The precipitating effecto f SDS was also examined at elevated temperatures. The two-phase systems of the proteins induced by SDS at 25 degrees C were heated from 25 degrees C to 90 degrees C at a rate of 1.25 degrees C/min. The precipitation behaviour was similar for the three proteins upon heating. When the SDS concentration was increased the precipitation curves were transferred towards lower temperatures and the courses of precipitation became less sharp. The synergistic effect of SDS and heat on protein precipitation was differentiated by denaturation measurements and radioactive labelling. The ratio SDS to precipitated protein gradually diminished towards higher temperatures but no purely thermal precipitation was found.     

Eosinophil transendothelial migration induced by cytokines. I. Role of endothelial and eosinophil adhesion molecules in IL-1 beta-induced transendothelial migration.

IL-1 beta promotes adhesiveness in human umbilical vein endothelial cells (HuVEC) for eosinophils through expression of adhesion molecules including intercellular adhesion molecules-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1). Using an in vitro endothelial monolayer system, we examined whether IL-1 beta or TNF-alpha can promote eosinophil transendothelial migration. We also evaluated the contributions of ICAM-1, E-selectin, VCAM-1, leukocyte adhesion complex (CD11/18), and very late Ag-4 (CD11b/18) (VLA-4) in this process using blocking mAb, and determined the changes in expression of CD11b and L-selectin on eosinophils that had undergone transmigration. IL-1 beta and TNF-alpha treatment of HuVEC (4 h, 5 ng/ml) induced significant transendothelial migration of eosinophils (a 4.1 +/- 0.4-fold (IL-1 beta) and 2.0 +/- 0.9-fold (TNF-alpha) increase from the spontaneous value of 3.2 +/- 0.3%). Increased CD11b expression and shedding of L-selectin were observed on eosinophils following IL-1 beta-induced eosinophil transendothelial migration. Studies with mAb revealed that blockade of either ICAM-1 or CD11/18 inhibited transmigration, while antibodies against VCAM-1 and VLA-4 had no inhibitory effect. Among antibodies which block beta 2 integrins, anti-CD18 mAb had the best inhibitory effect (88% inhibition). The combined inhibitory effect of anti-CD11a mAb and anti-CD11b mAb was roughly equal to that of anti-CD18, although anti-CD11a (31% inhibition) and anti-CD11b (52% inhibition) were less effective individually. Anti-ICAM-1 by itself inhibited IL-1 beta-induced eosinophil transendothelial migration (24% inhibition) whereas neither anti-E-selectin nor anti-VCAM-1 were effective inhibitors. Interestingly, the combination of anti-E-selectin and anti-VCAM-1 with anti-ICAM-1 inhibited IL-1 beta-induced eosinophil transendothelial migration significantly better (53% inhibition) than anti-ICAM-1 alone. These results suggest that although the initial attachment of eosinophils to IL-1 beta-activated endothelial cells involves VCAM-1, E-selectin, and ICAM-1, the subsequent transendothelial migration process relies heavily on ICAM-1 and CD11/18. Finally, the changes that eosinophils have been observed to undergo during infiltration in vivo, namely increased expression of CD11/18 and shedding of L-selectin, appear to take place as a direct result of the interaction between eosinophils and endothelial cells.     

Localization of eosinophil cationic protein, major basic protein, and eosinophil peroxidase in human eosinophils by immunoelectron microscopic technique

An immunoelectron microscopic technique using protein A-gold as a specific marker was used for precise intracellular localization of eosinophil granule proteins. Eosinophils from healthy individuals were isolated in metrizamide gradients. Eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) were clearly located in the matrix of the large crystalloid-containing granules. In addition, ECP was probably present in the small granules of eosinophils. Major basic protein (MBP) was present in the crystalloid structure of specific granules. This method can be applied in studies of eosinophil degranulation to trace the release of biological effector molecules.     

Visualization of Gluten and Starch Distributions in Dough by Fluorescence Fingerprint Imaging

A novel method combining imaging techniques and fluorescence fingerprint (FF) data measurement was developed to visualize the distributions of gluten and starch in dough without any preprocessing. Fluorescence images of thin sections of gluten, starch, and dough were acquired under 63 different combinations of excitation and emission wavelengths, resulting in a set of data consisting of the FF data for each pixel. Cosine similarity values between the FF of each pixel in the dough and those of gluten and starch were calculated. Each pixel was colored according to the cosine similarity value to obtain a pseudo-color image showing the distributions of gluten and starch. The dough sample was then fluorescently stained for gluten and starch. The stained image showed patterns similar to the pseudo-color FF image, validating the effectiveness of the FF imaging method. The method proved to be a powerful visualization tool, applicable in fields other than food technology.     

Identification of hematoxylin-stainable protein in epidermal keratohyalin granules as phosphorylated cystatin alpha by protein kinase C.

The hematoxylin-stainable protein (HSP) in keratohyalin granules of the newborn rat epidermis was found to have the same amino acid composition and the same inhibitory and immunological properties as cystatin alpha. However, only its pI value (4.7) differed from that of cystatin alpha (5.3). Alkaline phosphatase treatment of HSP changed its pI value from 4.7 to 5.3. This pI change was inhibited by EDTA, an inhibitor of alkaline phosphatase. Furthermore, 32P from [gamma-32P]ATP was incorporated into recombinant cystatin alpha by a protein kinase C (PKC) preparation in the presence of phosphatidyl serine and Ca2+ ions as co-factors. The incorporation increased dose-dependently with the added cystatin alpha and was inhibited significantly by H-7, a specific inhibitor of PKC. SDS-PAGE autoradiography of the 32P-labeled proteins showed that 32P was incorporated into the cystatin alpha. This incorporation was not observed by the action of cAMP-dependent protein kinase. Therefore, it is highly possible that the HSP is a phosphorylated cystatin alpha and that the phosphorylation is catalyzed specifically by PKC.     

A releationship between protein-degradation rates in vivo, isoelectric points, and molecular weights obtained by using density labelling.

1. Half-lives of five plant enzymes were estimated by rate-labeling with 2H2O on the assumption that loss of catalytic activity is equivalent to protein degradation. 2. This involved measuring band-broadening of activity profiles after isopycnic centrifugation. 3. Isoelectric points were determined by isoelectric focusing, and molecular weights were estimated by gel filtration. 4. The conclusion is drawn from the experimental evidence presented that a weak correlation exists between rates of degradation and isoelectric points (r = 0.699; P greater than 0.10; not significant). 5. A highly significant relationship exists between the logarithm of subunit size and half-life (r = -0.939; P greater than 0.02). 6. A literature survey confirmed the trends observed.     

Two-step purification of Cyanophora ferredoxin and its identification in soluble protein preparations by isoelectric focusing.

Cyanophora paradoxa ferredoxin is encoded by (cyano-)plastidic DNA, in contrast to those of all other photosynthetic eukaryotes investigated so far. In the present study we report (i) the rapid purification of a chloroplast-type [2Fe-2S] ferredoxin in a two-step procedure by DEAE-Sephadex and Mono Q ion-exchange chromatography; (ii) the biochemical characterization of the purified ferredoxin by electrophoretic separation methods on a microscale; and (iii) a qualitative and quantitative ferredoxin detection method in the femtomole range that allows densitometry, semidry immunoblotting, identification of ferredoxin in soluble cell protein preparations, and analysis of protein biosynthesis from cyanoplast poly(A)- RNA in vivo and in vitro. These fast micromethods should be useful for screening phototrophic species containing ferredoxins encoded by nonnuclear DNA.     

A comparison of the polypeptide isoelectric points and antigenic determinant sites of the large subunit of fraction 1 protein from Lycopersicon esculentum, Nicotiana tabacum and Petunia hybrida.

The large subunit of Fraction 1 protein from Lycopersicon esculentum, Nicotiana tabacum and Petunia hybrida has been examined by isoelectric focusing of the S-carboxymethylated polypeptides, and by double immunodiffusion with antiserum raised against Fraction 1 protein. The immunological results reveal heterogeneity in the large subunit primary structure not identified by isoelectric focusing. A variable phylogeny can be generated depending on whether serological or electrofocusing criteria are used.