Direct photo-induced DNA strand scission by a ruthenium bipyridyl complex

Irradiation of plasmid DNA in the presence of Ru(II)-2, a modified tris(2,2'-bipyridyl)Ru(II) complex, in which two hydroxamic acid groups are attached to one of the three bipyridyl ligands, results in total fragmentation of the DNA. The photo-chemical reaction products were analyzed by gel electrophoresis, which revealed complete fragmentation. Further evidence for the complete degradation of the DNA was obtained by imaging the pre- and post-treated plasmid DNA using atomic force microscopy (AFM). A mechanism for the reaction is proposed. It initially involves the photo-chemical generation of Ru(III) ions and superoxide radicals, as corroborated by absorbance difference spectroscopy and electron paramagnetic resonance (EPR). Consequently, Ru(III) preferentially oxidizes guanine, liberating superoxide radicals that yield OH radicals. The OH radicals were identified by observing the spectral change at 532 nm of a 5'-dAdG substrate forming a colored adduct with thiobarbituric acid. These radicals are associated with the major non-specific damage exerted to DNA.     

Fine structure of fusion products from soybean cell culture and pea leaf protoplasts

Protoplasts from pea (Pisum sativum L.) leaves and cultured soybean (Glycine max L.) cells were fused by means of polyethylene glycol and subsequently cultured for one week. Both agglutinated protoplasts and cultured fusion products were examined by electron microscopy. Agglutination occurred over large areas of the plasma membranes. The membrane contanct was discontinuous and irregularly spaced. Many cultured fusion products regenerated cell walls and divided to form cell clusters. Fusion of pea and soybean interphase nuclei occurred in some cells. The detection of heterochromatin typical of pea in the synkaryon, even after division, suggests the cells were hybrids. The cytoplasm of the cells from the fusion products contained both soybean leucoplasts and pea chloroplasts. The chloroplasts had apparently ceased dividing and some showed signs of degenerating. Large multinucleate fusion products developed cell walls but failed to divide.Abbreviations PEG polyethylene glycol - SEM scanning electron microscopy - TEM transmission electron microscopy Supported by National Research Council of Canada, Grant A6304     

The use of an inexpensive,general purpose microcomputer in quantitative cytochemistry

Summary The histochemical localization of alkaline phosphatase in sheep parathyroid gland was investigated using light and electron microscopy. The reaction products of enzyme activity were observed by light microscopy in pericytes. By electron microscopy they were limited to the intercellular spaces between the gland cells, being exclusively confined to the external surface of plasma membrane.     

Radiation- and chemical-induced structural chromosome aberrations in human spermatozoa

An apoptotic phenotype induced by oxygen radicals or Bax expression has been observed in Saccharomyces cerevisiae yeast cells by electron and fluorescence microscopy. In this work, we analyzed DNA content and cellular morphology of S. cerevisiae after H(2)O(2) or UV treatment by TdT-mediated dUTP nick end labeling (TUNEL)-test and flow cytofluorimetry. A TUNEL-positive phenotype was observed in both cases, on the same samples a dose-dependent increase in the sub-G(1) population was pointed out by flow cytometry. Sub-G(1) cells were isolated by flow sorting and analyzed by electron microscopy. This population showed condensed chromatin in the nucleus and cell shrinking. This paper reports the first evidence of apoptosis in yeast cells induced by DNA damage after UV irradiation.     

Effects induced by hydroxyl radicals on salmon calcitonin: a RP-HPLC,CD and TEM study

The effects of hydroxyl radical attack on a peptidic drug were studied in vitro. Different chemico-physical techniques were used to investigate structural damage induced by oxidative stress conditions in salmon calcitonin (sCT), a peptide hormone used in treating osteoporosis. Reversed-phase liquid chromatography (RP-HPLC), circular dichroism (CD) and transmission electron microscopy (TEM) were applied to measure formation of oxidation/degradation products and to reveal the conformational and ultrastructural modifications in the presence of OH. free radicals. Hydroxyl radicals were obtained from ferrous sulfate and ascorbic acid mixtures. The RP-HPLC results revealed the formation of new chromatographic peaks indicating a number of degradation/oxidation products formed in the presence of OH. free radicals. CD spectra showed slight protein conformational modifications as well as aggregation. TEM confirmed sCT aggregation and suggested the formation of fibrillar aggregates.     

Radical formation during the peroxidase catalyzed metabolism of carcinogens and xenobiotics: the reactivity of these radicals with GSH, DNA, and unsaturated lipid

Radicals generated by the peroxidase catalyzed oxidation of a wide variety of substrates oxidize GSH, NADH, or arachidonate with accompanying oxygen activation. Substrates studied include carcinogens, drugs, or xenobiotics. The effectiveness of the various radicals is partly related to their one-electron oxidation potential. High redox potential radicals were particularly effective at oxidizing these biomolecules. Low redox potential radicals did not react with GSH, NADH, or arachidonate, but can directly activate oxygen to form hydroxyl radicals or undergo scission to carbon radicals. The hydroxyl and carbon radicals have a high redox potential and readily oxidize biomolecules. DNA strand breakage also occurs with some high redox potential radicals, but DNA did not react with low redox potential radicals. The extensive binding of xenobiotics to DNA in the peroxidase system was attributed to noncovalent binding by polymeric products or covalent binding by the two electron oxidation product (formed by radical dismutation or oxidation). The latter can cause alkali labile DNA strand breaks. GSH conjugate formation was also attributed to the two electron oxidation product. Radicals have been trapped in intact cells and oxygen activation or lipid peroxidation has been demonstrated but it is still not clear whether the associated GSH oxidation, DNA strand breakage and cytotoxicity is the result of direct action by radicals. Indirect enzymic mechanisms for free radical mediated DNA strand breakage and cytotoxicity are discussed.     

“O_S-732人体成骨肉瘤细胞系”超微结构及细胞化学的研究

本文研究了 Os-732细胞株的超微结构及酶的特性。在 SEM 下细胞形态呈多样性,细胞表面形成树枝状伪足;TEM 下胞质内含丰富的扩张粗面内质网,发达的高尔基复合体及分泌泡;大量碱性磷酸酶作用的沉积物出现于胞膜表面。这些特征均显示它与成骨细胞的相似性。为证明此株细胞是威骨系统的肿瘤提供可靠的依据。结合细胞株的动物接种,染色体和免疫学特性等研究,证明此细胞株是一新的成骨肉瘤细胞株。     

Effects of X-irradiated Aqueous Solutions on Vesicular Stomatitis Virus

Infectivity of vesicular stomatitis virus was inactivated by suspending the virus in aqueous salt solutions that had previously been subjected to X-irradiation. This viral inactivation did not appear to be attributable to hydrogen peroxide or to long-lived free radicals generated by X-irradiation of the salt solutions. Indirect evidence suggested that the deleterious effects on the virus were caused by chemical species resulting from interaction of free water molecules with anions in the solution. Inactivation of infectivity was associated with distortion of the outer layer of the virion, as determined by electron microscopy, and with consequent impairment of adsorption to otherwise susceptible cells.     

Radicalization of lignocellulosic fibers, related structural and morphological changes

The radicalization of unbleached lignocellulosic fibers obtained from thermomechanical (TMP) and chemothermomechanical (CTMP) pulps was performed in heterogeneous phase by reaction with dioxygen in the presence of N,N'-ethylenebis(salicylideneiminato)cobalt(II), [Co(salen)], as catalyst. Phenoxy cobalt radicals immobilized in fibers were observed by electron paramagnetic resonance (EPR) spectroscopy; their amount depends on the fiber swelling induced by reaction medium. The absolute concentration of such radicals in fibers, about 10(16) spin/g, reaches values 10 times higher than that of phenoxy radicals formed in similar oxidative reactions catalyzed by laccase. The generation of phenoxy cobalt radicals in fibers was related to structural changes of lignin units, detected by mono- and bidimensional nuclear magnetic resonance ((13)C NMR and 2D-HSQC) investigations, and to morphological modifications in fibers observed by scanning electron microscopy (SEM).     

Cell cycle and morphological alterations as indicative of apoptosis promoted by UV irradiation in S. cerevisiae

An apoptotic phenotype induced by oxygen radicals or Bax expression has been observed in Saccharomyces cerevisiae yeast cells by electron and fluorescence microscopy. In this work, we analyzed DNA content and cellular morphology of S. cerevisiae after H₂O₂ or UV treatment by TdT-mediated dUTP nick end labeling (TUNEL)-test and flow cytofluorimetry. A TUNEL-positive phenotype was observed in both cases, on the same samples a dose-dependent increase in the sub-G₁ population was pointed out by flow cytometry. Sub-G₁ cells were isolated by flow sorting and analyzed by electron microscopy. This population showed condensed chromatin in the nucleus and cell shrinking. This paper reports the first evidence of apoptosis in yeast cells induced by DNA damage after UV irradiation.     

Cell-mediated reduction of protein and peptide hydroperoxides to reactive free radicals

Radical attack on proteins in the presence of O(2) gives protein hydroperoxides in high yields. These peroxides are decomposed by transition metal ions, reducing agents, UV light and heat, with the formation of a range of reactive radicals that are capable of initiating further damage. Evidence has been presented for the formation of alcohols as stable products of peroxide decomposition, and these have been employed as markers of oxidative damage in vivo. The mechanism of formation of these alcohols is unclear, with both radical and nonradical pathways capable of generating these products. In this study we have investigated the reduction of peptide and protein hydroperoxides by THP-1 (human monocyte-like) cells and it is shown that this process is accompanied by radical formation as detected by EPR spin trapping. The radicals detected, which are similar to those detected from metal-ion catalyzed reduction, are generated externally to the cell. In the absence of cells, or with cell-conditioned media or cell lysates, lower concentrations of radicals were detected, indicating that intact cells are required for rapid hydroperoxide decomposition. The rate of radical generation was enhanced by preloading the cells with ascorbate, and this was accompanied by intracellular formation of the ascorbate radical. It is proposed that decomposition of some amino acid and peptide hydroperoxides occurs extracellularly via the involvement of a cell-surface reducing system, such as a trans-plasma membrane electron transport system (TPMET) either directly, or indirectly via redox cycling of trace transition metal ions.     

Effects of high-pressure processing on in vitro infectivity of Encephalitozoon cuniculi

High-pressure processing (HPP) has been shown to be an effective means of eliminating bacteria and destructive enzymes from a variety of food products. HPP extends the shelf life of products while maintaining the sensory features of food and beverages. In this study, we examined the effects of HPP on the infectivity of Encephalitozoon cuniculi spores in vitro. Spores were exposed to between 140 and 550 MPa for 1 min in a commercial HPP unit. Following treatment, the spores were loaded onto cell culture flasks or were kept for examination by transmission electron microscopy. No effect was observed on the infectivity of spores treated with 140 MPa. Spores treated with between 200 and 275 MPa showed reduction in infectivity. Following treatment of 345 MPa or more, spores were unable to infect host cells. No morphologic changes were observed in pressure-treated spores with transmission electron microscopy.     

Diaziquone as a potential agent for photoirradiation therapy: formation of the semiquinone and hydroxyl radicals by visible light

When diaziquone was irradiated with 500 nm visible light, hydroxyl free radicals as well as the diaziquone semiquinone were produced. The diaziquone semiquinone is a stable free radical that exhibits a characteristic 5-line electron spin resonance (ESR) spectrum. Since hydroxyl free radicals are short lived, and not observable by conventional ESR, the nitrone spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) was used to convert hydroxyl radicals into longer lived ESR detectable spin adducts. The formation of hydroxyl radicals was further confirmed by investigating reactions in which hydroxyl radical scavangers, sodium formate and dimethylsulfoxide, compete with the spin traps DMPO or POBN (alpha-(4-Pyridyl-1-oxide)-N- tert-butylnitrone) for hydroxyl free radicals. The products of these scavenging reactions were also trapped with DMPO or POBN. If drug free radicals and hydroxyl free radicals are important in the activity of quinone-containing antitumor agents, AZQ may have a potential in photoirradiation therapy or photodynamic therapy.     

Pulse radiolysis study of the interaction of retinoids with peroxyl radicals

Vitamin A (retinol) and its derivatives-retinal and retinoic acid-are known for their ability to inhibit lipid peroxidation. Antioxidant actions of retinoids have been attributed to chain-breaking by scavenging of peroxyl radicals. Based on chemical analysis of retinoic acid degradation products formed during microsomal lipid peroxidation, it was previously suggested that retinoids interact with peroxyl radicals forming free carbon-centered radical adducts. However, it can be argued that such a mode of antioxidant action of retinoids is not sufficient to fully explain their effectiveness at inhibiting lipid peroxidation, which in many systems is comparable to, or even exceeds, that of alpha-tocopherol. In order to elucidate the mechanism of interaction of retinoids with peroxyl radicals, (trichloromethyl)peroxyl radical was generated by pulse radiolysis, and its interactions with retinoids solubilized in Triton X-100 micelles were followed by kinetic absorption spectroscopy. All retinoids--retinol, retinal, and retinoic acid--interacted with the peroxyl radical, and at least two transient products were detected. One of these products, absorbing at 590 nm, was identified as retinoid cation radical. Therefore, we postulate that, apart from formation of radical adducts, retinoids may also scavenge peroxyl radicals by electron transfer.     

Ultrastructure of mast cell degranulation induced by eosinophil peroxidase: Use of diaminobenzidine cytochemistry by scanning electron microscopy

It has been previously demonstrated that eosinophil peroxidase (EPO) when supplemented with hydrogen peroxide and a halide induces noncytotoxic mast cell degranulation. Using a more highly purified EPO preparation, the ultrastructure of EPO-induced mast cell secretion has been studied using transmission and scanning electron microscopy and freeze-fracture techniques. At relatively low EPO concentrations, secretory changes were comparable to those caused by other mast cell secretagogues. Swollen and less electron-dense granules were seen in intracellular channels, some of which opened to the outside of the cell. EPO stimulation led to bulging of the surface membrane by submembranous granules and formation of pores in the cell surface that also contained fewer villous projections than control cells. During the secretory process, plasma membrane bulges were depleted of intramembranous particles in both the E and P faces of the apical regions of the perigranular and plasma membranes. Higher EPO concentrations caused a marked cytotoxic disruption of the mast cells. Diaminobenzidine cytochemistry was used to detect EPO reaction products on the mast cell surface by scanning electron microscopy; this technique should prove useful in detecting peroxidase reaction products on a variety of target cells.     

Histochemical and cytochemical localization of (Na+-K+)-activated adenosine triphosphatase in the acini of dog submandibular glands.

p-Nitrophenyl phosphatase (p-NPPase) activity of (Na+-K+)-activated adenosine triphosphatase ((Na+-K+)-ATPase) on the acinar cells of dog submandibular gland was demonstrated by using light microscopy. The reaction products of p-NPPase of fresh frozen sections were seen to be localized on the basal parts of acini, and disappeared when the sections were incubated in medium containing 10(-3) Mouabain or in a K-free medium. Under the electron microscope, the reaction products of ATPase were found to be localized on the basolateral plasma membrane of both serous and mucous cells. On the microvilli of the luminal plasma membrane of the acinar cell, a small quantity of the reaction products was also present. This localization of ATPase reaction products on the serous and mucous cells seemed to coincide well with that of p-NPPase activity observed on the acini under light microscopy. Possible explanations are given regarding distribution of the above mentioned enzymes in relation to the cation transport of the plasma membrane. Structural and functional asymmetrical properties of acinar cells of the dog submandibular gland are also discussed.     

Mast cell dynamics in the house rat (Rattus rattus) ovary during estrus cycle, pregnancy and lactation.

The distribution of mast cells in various ovarian compartments was studied during different stages of the reproductive cycles in Rattus rattus. Two types of mast cell populations were recognized with light microscopy i.e., light purple and deep purple, the latter also includes deeply stained cells with extruded granules. Mast cells identified by electron microscopy showed the ultrastructural features during granule formation and release of their content. Significantly higher numbers of mast cells per unit area of ovary were seen at estrus and diestrus. Numbers of mast cells also remained high during pregnancy with possible involvement of mast cell products in vascularization of corpora lutea. A positive correlation existed between mast cell counts and embryo number during pregnancy. However, numbers of mast cells declined significantly after parturition.     

Ultrastructure of cells cultured on polycarbonate membranes.

A method is described for preparing undisturbed cell cultures for both scanning and transmission electron microscopy. Cells were propagated on polycarbonate membranes with pores of 0.2 micrometer or less. Cultured cells together with their supports were prepared for both scanning electron microscopy and transmission electron microscopy using routine methods. For transmission electron microscopy a rapid schedule of infiltration and polymerization was used. The method described in this report yielded good results and it allowed the fine structure of cultured cells to be viewed in situ by both scanning electron microscopy and transmission electron microscopy.     

Electron and hole transfer from DNA base radicals to oxidized products of guanine in DNA

An investigation of electron and hole transfer to oxidized guanine bases in DNA is reported. Guanine in DNA was preferentially oxidized by Br(2)(*-) at 298 K to 8-oxo-7,8-dihydro-guanine (8-oxo-G) and higher oxidation products. HPLC-EC analysis of irradiated DNA shows that the formation of 8-oxo-G could not be increased above the ratio of one 8-oxo-G to 127 +/- 6 bp regardless of dose. 8-oxo-G can be produced only at low levels because it is further oxidized to other species. These oxidation products of guanine have been extensively investigated and identified by others. Our electron spin resonance studies suggest that at 77 K 8-oxo-G is a trap for radiation-produced holes, but certain further oxidation products of 8-oxo-G (G(ox)) are found to be efficient electron traps. Gamma irradiation of oxidized DNA samples in frozen (D(2)O) aqueous ices and glassy 7 M LiBr solutions resulted in radicals formed by electron attachment to the G(ox) sites that were monitored by electron spin resonance spectroscopy (ESR) at 77 K. These ESR spectra suggest that those oxidation products of 8-oxo-G containing alpha-diketo groups account for the electron traps (G(ox)) in oxidized DNA with oxaluric acid a likely major trap. Electron transfer from DNA anion radicals to G(ox) was followed by monitoring of their ESR signals with time at 77 K. Using typical values for the tunneling constant beta estimates of the relative amount of G(ox) to base pairs were obtained. Radicals formed by UV photolysis of oxidized DNA in 8 M NaClO(4) glassy aqueous solutions were also investigated. The 8-oxo-G cation accounts for less than 10% of all the radicals observed after either gamma irradiation of oxidized DNA in frozen (D(2)O) aqueous solution or UV photolysis of oxidized DNA in 8 M NaClO(4) glassy aqueous solutions. We estimate hole transfer distances of about 7 +/- 1 bp at 1 min from G(*+) to 8-oxo-G.     

Ultrastructure of Cells Cultured on Polycarbonate Membranes

A method is described for preparing undisturbed cell cultures for both scanning and transmission electron microscopy. Cells were propagated on polycarbonate membranes with pores of 0.2 pm or less. Cultured cells together with their supports were prepared for both scanning electron microscopy and transmission electron microscopy using routine methods. For transmission electron microscopy a rapid schedule of infiltration and polymerization was used. The method described in this report yielded good results and it allowed the fine structure of cultured cells to be viewed in situ by both scanning electron microscopy and transmission electron microscopy.