Light generation with Fenton's reagent. Its relationship to granulocyte chemiluminescence.

A simple chemical system consisting of FeSO4 and H2O2 (Fenton's reagent) was shown to emit light (chemiluminescence). The addition of tryptophan to the reaction markedly enhanced light production. Very little chemiluminescence was observed when H2O2 was omitted from the reaction and when ferric, instead of ferrous, ions were used. Hydroxyl radical (OH.) and singlet oxygen (1 deltagO2) quenchers suppressed chemiluminescence of the FeSO4 + tryptophan + H2O2 system; and, deuterium oxide (2H2O) enhanced chemiluminescence of both FeSO4 reactions. These observations suggest that a radical chain reaction involving both OH. and 1 deltag O2 is responsible for the chemiluminescent reactions. Six iron-containing proteins, some of which are located within granulocytes, all emitted light in the presence of H2O2. Since iron and H2O2 are present in metabolically stimulated granulocytes, it is likely that chemiluminescent reactions similar to the ones demonstrated in this study account for part of the chemiluminescence of activated granulocytes.     

The effect of buffers and chelators on the reaction of luminol with Fenton's reagent near neutral pH

The chemiluminescence of the system luminol +Fe²⁺ + H₂O₂ was measured in aqueous buffer at pH 7.2. In veronal (5,5-diethybarbiturate) buffer, the luminescence is strongly quenched by ethanol and mannitol, but only weakly by t-butanol, benzoate and superoxide dismutase (SOD); complexing Fe²⁺ with 1,10-phenanthroline or 2,2′-dipyridyl causes a decrease of light production that can be partially obviated by the simultaneous addition of SOD. In phosphate buffer, the luminescence is higher than in veronal and it is efficiently quenched by all four OH · quenchers and by SOD. In Tris buffer, no light production is observed as long as the Fe²⁺ is not complexed. When Fe²⁺ is complexed by pyrophosphate or phytate, there is a strong chemiluminescence in all three buffers, which is quenched by all four OH · quenchers and by SOD. When Fe²⁺ is complexed by EDTA or DTPA, very little luminescence is observed. The luminol analogue phthalhydrazide, which was suggested by Merényi and Lind as a reliable OH · detector, can replace luminol only in phosphate buffer, and thus turns out to be very specific indeed for free OH ·.     

Correlation between hydroperoxide-induced chemiluminescence of the heart and its function

The isolated perfused rat heart emits a spontaneous ultraweak chemiluminescence. When the perfusion is stopped, light emission decreases, indicating the dependency of this phenomenon on aerobic metabolism. Emitted chemiluminescence was markedly enhanced following perfusion with 0.05 mM H₂O₂ or cumene hydroperoxide or tert-butyl hydroperoxide; substitution of O₂ for N₂ in the gassing mixture of the perfusion media significantly lowered photon emission. Lipid peroxidation, which is known to be associated with chemiluminescence, was evaluated by HPLC analysis of peroxidized and unperoxidized heart phosphatidylcholines. During hydroperoxide perfusion, coronary flow and heart rate progressively decreased, while lactic dehydrogenase was released after complete cardiac arrest. The resultant morphology of this damage corresponds to the so-called ‘stone heart’, a pattern already described in both human and experimental pathology.     

Abnormalities of lysosomes in human diploid fibroblasts from patients with Farber's disease

An accumulation of ceramide associated with the deficiency of acid ceramidase has been demonstrated in cultured diploid skin fibroblasts from a patient with Farber's disease. We extend this observation to investigate the lysosomal localization of accumulated ceramide and the abnormalities of lysosomes caused by this ceramide accumulation in Farber's diseased fibroblasts. We have found that the lysosomal fraction isolated from Farber's diseased fibroblasts by a subcellular fractionation procedure is markedly low in density compared with that of normal fibroblasts and is separated from other subcellular organellers. Ultrastructural studies of the isolated lysosomal fraction from Farber's diseased fibroblasts showed in mixed population of intact and swollen vesicles with a lysosomal appearance. Examination under high magnification clearly demonstrated lysosomal inclusions which contain lamellar and curvilinear membranes and resembled those seen in the intact fibroblasts. Subcellular localization of Farber's fibroblasts showed that the accumulated [³H]ceramide from the culture medium was predominantly localized in the lysosomal fraction with a markedly low density and very little was found to be associated with other cellular membranes. Our finding that ceramide is accumulated in the lysosomal fraction of Farber's fibroblasts and that these cells also show membranous inclusions strongly suggests that the accumulation of ceramide is directly involved in the formation of lysosomal inclusions.     

The differential reaction of histamine and N tau-methylhistamine with Pauly's diazo reagent: application to assay of histamine N-methyltransferase activity

A simple nonradioisotopic fluorescent method for assay of histamine N-methyltransferase (HMT) activity was developed. After termination of the HMT reaction, the remaining excess substrate, histamine, was degraded by Pauly 's diazo reagent, whereas the product, N tau-methylhistamine (N- MeHA ), was not degraded by the reagent. Then the mixture was applied to high-performance liquid chromatography under conditions in which N- MeHA was not separated from histamine, and N- MeHA was measured fluorometrically by condensation with o-phthalaldehyde. The method would be convenient for measurement of HMT activity during enzyme purification.     

In vitro screening of Fe2+‐chelating effect by a Fenton's reaction–luminol chemiluminescence system

In vitro screening of a Fe²⁺‐chelating effect using a Fenton's reaction–luminol chemiluminescence (CL) system is described. The luminescence between the reactive oxygen species generated by the Fenton's reaction and luminol was decreased on capturing Fe²⁺ using a chelator. The proposed method can prevent the consumption of expensive seed compounds (drug discovery candidates) owing to the high sensitivity of CL detection. Therefore, the assay could be performed using small volumes of sample solution (150 μL) at micromolar concentrations. After optimization of the screening conditions, the efficacies of conventional chelators such as ethylenediaminetetraacetic acid (EDTA), diethylentriaminepentaacetic acid (DETAPAC), deferoxamine, deferiprone and 1,10‐phenanthroline were examined. EC₅₀ values for these compounds (except 1,10‐phenanthroline) were in the range 3.20 ± 0.87 to 9.57 ± 0.64 μM (n = 3). Rapid measurement of the Fe²⁺‐chelating effect with an assay run time of a few minutes could be achieved using the proposed method. In addition, the specificity of the method was discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Molecular heterogeneity in McArdle's disease

Biopsies were taken from a group of eleven patients with McArdle's disease, a congenital deficiency in muscle glycogen phosphorylase. The biopsies were screened by Western and Northern blotting for phosphorylase protein, phosphotrylase-bound pyridoxal-5′-phosphate (the cofactor of the enzyme) and for phosphorylase mRNA. Of the eleven patients, three expressed phosphorylase mRNA at near normal levels and at the expected size. One of these patients also expressed low levels of phosphorylase protein that correlated with a small amount of measurable phosphorylase activity. These data support the contention of molecular heterogeneity in the presentation of this phenotype.     

Synthesis, crystal structure and characterizations of iron(III) and copper(II) complexes with the hydrazone ligand obtained from 2-formyl-pyridine and Girard’s T reagent

The condensation of 2-formyl-pyridine with Girard’s T reagent yields a new hydrazone in the form of an ammonium quaternary salt: [H(2-PyGT)]Cl. This tridentate ligand is readily soluble in water and reacts with iron(III) or copper(II) chlorides to give [Fe(2-PyGT)Cl₃] (1) or [Cu(2-PyGT)Cl₂]·(H₂O) (2) complexes, respectively. Single-crystal X-ray studies in 1 and 2 reveal that the coordination reaction gives rise to the deprotonation of the organic ligand that is coordinated using its NNO donor atoms in the form of a zwitterion species. The coordination spheres around the transition metal ions in complexes 1 and 2 are quite different. In 1, the iron site adopts a distorted octahedral coordination sphere, while the Cu(II) ions in 2 show a distorted tetragonal-pyramid geometry. As expected, the magnetic properties of these compounds reveal only weak antiferromagnetic interaction between spin carriers.     

Approach to the mechanism of Zajdela's hepatoma cell growth inhibition induced by concanavalin A and phytohaemagglutinin

Cell growth of tumour ascites cell was inhibited by concanavalin A, phytohaemagglutinin and Ricinus lectin at 2–100 μg/ml. As expected, the Ricinus lectin inhibited the protein synthesis estimated by leucine incorporation and decreased thymidine incorporation, whereas concanavalin A and phytohaemagglutinin stimulate the uptake and the incorporation of both leucine and thymidine, and thus, synthesis of protein and DNA. Theses results suggest that different mechanisms are involved in the hepatoma cell growth inhibition by the lectins. This difference was not related to the kinetic characteristics of the lectin interactions with the cells whihc represent a first and necessary step. It was showed that concanavalin A and phytohaemagglutinin as well as chloroquine inhibited the ¹⁴C-labelled asialofetuin degradation. We can conclude that Ricinus lectic present a toxic effect whereas both concanavalin A and phytohaemagglutinin show an anti-protease activity.     

Identification of damaged proteins in human serum using modified Ehrlich’s reagent to target protein-bound pyrroles

Protein-bound pyrroles are a sign of oxidative damage. Here we report a specific method for detecting pyrrole-containing proteins using biotin-labeled Ehrlich’s reagent (ER-B). After treatment of either human serum or isolated human serum proteins with various oxidizing agents, damaged, biotin-labeled components could be detected by blotting. Combining the use of ER-B with proteomic techniques allowed human serum proteins susceptible to oxidative damage to be detected and then identified by LC/MS/MS. Identification of such proteins in different human conditions such as obesity, diabetes, and cardiovascular disease should lead to the discovery of new biomarkers and the development of specific assays to monitor health status.     

Enhanced chemiluminescence: A sensitive analytical system for detection of sweet potato peroxidase

3-(10'-Phenothiazinyl)propane-1-sulfonate (SPTZ) was shown to be a potent enhancer of anionic sweet potato peroxidase (aSPP)-induced chemiluminescence. The optimal conditions for aSPP-catalyzed oxidation of luminol were investigated by varying the concentrations of luminol, hydrogen peroxide, Tris, and SPTZ as well as the pH values of the reaction mixture. Addition of 4-morpholinopyridine (MORP) to the reaction mixture markedly increased the light intensity. Using SPTZ and MORP together enhanced the effect 265 times. The lower detection limit (LDL) of SPP was 0.09 pM, approximately in 10 times lower than that for the cationic isozyme c of horseradish peroxidase/4-iodophenol system. It was shown that aSPP in the presence of SPTZ produced a longer lasting chemiluminescent signal.     

Engineering superactive granulocyte macrophage colony‐stimulating factor transferrin fusion proteins as orally‐delivered candidate agents for treating neurodegenerative disease

Intravenously injected granulocyte macrophage colony‐stimulating factor (GM‐CSF) has shown efficacy in Alzheimer's Disease (AD) and Parkinson's Disease (PD) animal studies and is undergoing clinical evaluation. The likely need for dosing of GM‐CSF to patients over months or years motivates pursuit of avenues for delivering GM‐CSF to circulation via oral administration. Flow cytometric screening of 37 yeast‐displayed GM‐CSF saturation mutant libraries revealed residues P12, H15, R23, R24, and K72 as key determinants of GM‐CSF's CD116 and CD131 GM‐CSF receptor (GM‐CSFR) subunit binding affinity. Screening combinatorial GM‐CSF libraries mutated at positions P12, H15, and R23 yielded variants with increased affinities toward both CD116 and CD131. Genetic fusion of GM‐CSF to human transferrin (Trf), a strategy that enables oral delivery of other biopharmaceuticals in animals, yielded bioactive wild type and variant cytokines upon secretion from cultured Human Embryonic Kidney cells. Surface plasmon resonance (SPR) measurements showed that all evaluated variants possess decreases in CD116 and CD131 binding K_D values of up to 2.5‐fold relative to wild type. Improved affinity led to increased in vitro bioactivity; the most bioactive variant, P12D/H15L/R23L, had a leukocyte proliferation assay EC₅₀ value 3.5‐fold lower than the wild type GM‐CSF/Trf fusion. These outcomes are important first steps toward our goal of developing GM‐CSF/Trf fusions as orally available AD and PD therapeutics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:668–677, 2015     

Electrogenerated chemiluminescence of tris(2‐phenylpyridine)iridium(III) in water,acetonitrile and trifluorethanol

The spectroscopic, electrochemical and coreactant electrogenerated chemiluminescence (ECL) properties of Ir(ppy)3 (where ppy = 2‐phenylpyridine) have been obtained in aqueous buffered (KH2PO4), 50 : 50 (v/v) acetonitrile–aqueous buffered (MeCN–KH2PO4) and 30% trifluoroethanol (TFE) solutions. Tri‐n‐propylamine was used as the oxidative‐reductive ECL coreactant. The photoluminescence (PL) efficiency (ϕem) of Ir(ppy)3 in TFE (ϕem ≈ 0.029) was slightly higher than in 50 : 50 MeCN–KH2PO4 (ϕem ≈ 0.0021) and water (ϕem ≈ 0.00016) compared to a Ru(bpy)32+ standard solution in water (Φem ≈ 0.042). PL and ECL emission spectra were nearly identical in all three solvents, with dual emission maxima at 510 and 530 nm. The similarity between the ECL and PL spectra indicate that the same excited state is probably formed in both experiments. ECL efficiencies (ϕecl) in 30% TFE solution (ϕecl = 0.0098) were higher than aqueous solution (ϕecl = 0.00092) system yet lower than a 50% MeCN–KH2PO4 solution (ϕecl = 0.0091). Copyright © 2014 John Wiley & Sons, Ltd.     

Normal response of Fanconi's anemia cells to high concentrations of O2 as determined by alkaline elution

In this investigation, normal and Fanconi's anemia fibroblasts were exposed to high concentrations of oxygen and the effects of this treatment on DNA were analyzed by alkaline elution. No DNA single-strand breaks were detected in either cell type with up to 20 h incubation in high (50–95%) concentrations of O₂. No evidence of DNA damage by O₂ could be detected with an endonuclease preparation from Micrococcus luteus. Cells which have been treated with various DNA-damaging agents in the presence of the polymerase inhibitor cytosine arabinoside have been shown to accumulate DNA single-strand breaks during DNA excision repair. When cells were treated with the polymerase inhibitor in 50 or 95% O₂, a low level of DNA single-strand breaks accumulated in both cell types. However, no significant differences in the frequency of DNA single-strand breaks were detected between normal and Fanconi's anemia cells after exposure to high O₂.     

X-ray diffraction evidence for myelin disorder in brain from humans with alzheimer's disease

Wide-angle X-ray diffraction studies revealed that the lipid phase transition temperature of myelin from brain tissue of humans with Alzheimer's disease was about 12°C lower than that of normal age-matched controls, indicating differences in the physical organization of the myelin lipid bilayer. Elevated levels of malondialdehyde and conjugated diene were found in brain tissue from humans with Alzheimer's disease, indicating an increased amount of lipid peroxidation over the controls. An increase in myelin disorder and in lipid peroxidation can both be correlated with aging in human brain, but the changes in myelin from humans with Alzheimer's disease are more pronounced than in normal aging. These changes might represent severe or accelerated aging.     

Evidence for a mechanical coupling of glycoprotein microfibrils with collagen fibrils in Wharton's jelly

Wharton's jelly of human umbilical cord is known to contain hyaluronic acid and sulphated glycosaminoglycans (probably as proteoglycans) immobilized in an insoluble collagen fibril network. A secondary, independent, insoluble network based on glycoprotein microfibrils of 13 nm diameter and interpenetrated with the collagen network has now been found in amounts corresponding to 9% of the weight of collagen. Elastin, however, is absent. Tissue slices placed in physiological buffer swell to two-fold their in vivo volume. This is due to the influence of the polysaccharides since treatment with either testicular hyaluronidase, Streptomyces hyaluronidase or chondroitinase ABC, causes their quantitative removal and abolishes the swelling tendency of tissue. Tissue so treated remains close to its in vivo volume indicating that for this state the fibrillar network, overall, is in its relaxed unstressed configuration. Subsequent treatment with a protease causes the degradation of the glycoprotein microfibril network and a two-fold increase in tissue volume while treatment with bacterial collagenase, resulting in the solubilization of 46% of the collagen, causes only a slight deswelling. These results suggest that the unstressed configuration of the network system at the in vivo volume of tissue is due to the collagen network being held in compression by the microfibril network. With intact tissue protease digestion with trypsin, in addition, causes a preferential release of sulphated glycosaminoglycans. Hyaluronic acid, however, remains largely immobilized.     

DNA fork displacement rates in Bloom's syndrome fibroblasts

DNA fork displacement rates were measured in three lines of Bloom's syndrome cells and in a normal diploid fibroblast line. Fork displacement rates in Bloom's cells were approx. 55–65% of the rate in normal fibroblasts.