Eimeria maxima: identification of gametocyte protein antigens

The antigenicity of Eimeria maxima gametocyte proteins during the course of an infection and when injected into mice and rabbits was demonstrated using the Western blotting technique. Serum taken from chickens at various times postinfection reacted to a few gametocyte proteins, with the strongest reactivity seen with serum taken 14-days postinfection. Two major antigens having molecular weights of 56,000 and 82,000 were consistently detected by these sera. Using immune rabbit or mouse sera to whole gametocyte detergent extracts, the 56,000 and 82,000 molecular weight proteins were again the immunodominant antigens, despite their representing only a small proportion of the extract which was used to immunize the animals. These results, together with those obtained by Rose (1971) using recovered chicken serum to passively immunize chickens, indicate that these two gametocyte antigens may play a role in protective immunity to E. maxima.     

Pure exogenous singlet oxygen: nonmutagenicity in bacteria

Singlet oxygen (1 delta gO2) is the lowest energy-excited state of molecular oxygen, and more reactive than the triplet ground-state molecule. Although singlet oxygen has been implicated in a variety of biological effects, including reactions with DNA or some of its components, evidence for mutagenesis by singlet oxygen has remained unclear. We have previously described a system for bacterial exposure to pure exogenous singlet oxygen that eliminates ambiguity regarding the identity of the reactive species responsible for observed results. Despite the potent toxicity of pure singlet oxygen for several different strains of bacteria, we have found no evidence for mutagenicity of singlet oxygen in 26 Salmonella typhimurium histidine-auxotrophic strains killed to 35% survival. These strains included a variety of base-pair substitution or frameshift target sequences for reversion, including targets responsive to oxidative damage and targets rich in GC base pairs. Some strains combined histidine mutations with one or more mutations affecting DNA-repair capacity. 4 strains possessing the hisG46 mutation also were not mutated when exposed to dose ranges killing less than 28% and up to 99% of the bacteria. The relative frequency of small inphase deletions was assayed in hisG428 bacteria exposed to single oxygen and found to be the same as the spontaneous level. In addition to lack of induction of mutation in these strains, the 8-azaguanine forward mutation assay yielded no evidence of mutagenesis by singlet oxygen in strains killed to 15% survival. No induction of genetic changes by singlet oxygen was seen in an assay for duplication of approximately 1/3 of the bacterial chromosome. Tests for the ability of singlet oxygen to induce lambda prophage in E. coli K12 also proved negative. These studies collectively indicate that pure singlet oxygen generated outside the bacterial cell does not react significantly with the bacterial chromosome in ways leading to base-pair substitutions, frameshift mutations, small or large deletions, large duplications, or damage that interferes with DNA replication and induces the SOS system.     

The relationship between serum nitrate and endothelin-1 concentrations in preeclampsia

Recently it was suggested that abnormal endothelial function may contribute to the pathophysiological changes observed in preeclampsia (PE). Both nitric oxide (NO) and endothelin-1 (ET-1) are vasoactive substances produced by endothelial cells. NO is a vasodilator and has been believed to be decreased in PE. ET-1 is a vasoconstrictor and has been reported to be increased in PE. We simultaneously measured NO metabolites and ET-1 in sera from women with PE and investigated the correlation of NO and ET-1 concentrations. We obtained serum samples from 11 healthy nonpregnant (NP) women, 16 normotensive pregnant (NTP) women and 17 women with PE. In this study, the serum ET-1 level was assayed by the ET-1 RIA system, and serum NO metabolites were assayed by measuring nitrite (NO2-) and nitrate (NO3-) simultaneously in an HPLC-Griess reaction system. There was a significant correlation between NOx (nitrite + nitrate) and ET-1 in sera from all 44 women (NP, NTP and PE groups) (p<0.001). Nitrite and ET- in sera from each group were not significantly correlated. Nitrate and ET-1 in sera from the NP and NTP groups did not significantly correlate. However, there was a significant correlation between nitrate and ET-1 in sera from the PE group (p<0.05). The serum ET-1 and nitrate concentration in the PE group was significantly higher than in the NP and NTP groups (p<0.05 and p<0.001. respectively). These findings suggest that increased production of nitrate in PE may contribute to homeostatic vasodilation against vasoconstriction caused by a higher ET-1 concentration.     

Inactivation of Neurospora crassa conidia by singlet molecular oxygen generated by a photosensitized reaction.

Photodynamic damage of Neurospora crassa conidia was studied in the presence of the photosensitizing dye, toluidine blue O. Conidia which germinated to form colonies decreased in number as irradiation time became longer. The photoinactivation of conidia was suppressed by azide, bovine serum albumin, and histidine, and was stimulated in deuterium oxide. Wild-type conidia were less sensitive to the irradiation than albino conidia. In the wild-type, carotenoid-enriched conidia were more resistant against the lethal damage than the conidia which contained small amounts of carotenoids. These results suggest that singlet molecular oxygen causes photodynamic lethal damage to N. crassa conidia and that singlet molecular oxygen is quenched by endogenous carotenoids.     

Control of singlet oxygen-induced oxidative damage in Escherichia coli

Singlet oxygen ((1)O(2)) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. The oxyR gene product regulates the expression of the enzymes and proteins that are needed for cellular protection against oxidative stress. In this study, the role of oxyR in cellular defense against a singlet oxygen was investigated using Escherichia coli oxyR mutant strains. Upon exposure to methylene blue and visible light, which generates singlet oxygen, the oxyR overexpression mutant was much more resistant to singlet oxygen-mediated cellular damage when compared to the oxyR deletion mutant in regard to growth kinetics, viability and protein oxidation. Induction and inactivation of major antioxidant enzymes, such as superoxide dismutase and catalase, were observed after their exposure to a singlet oxygen generating system in both oxyR strains. However, the oxyR overexpression mutant maintained significantly higher activities of antioxidant enzymes than did the oxyR deletion mutant. These results suggest that the oxyR regulon plays an important protective role in singlet oxygen-mediated cellular damage, presumably through the protection of antioxidant enzymes.     

Genetically targeted chromophore-assisted light inactivation

Studies of protein function would be facilitated by a general method to inactivate selected proteins in living cells noninvasively with high spatial and temporal precision. Chromophore-assisted light inactivation (CALI) uses photochemically generated, reactive oxygen species to inactivate proteins acutely, but its use has been limited by the need to microinject dye-labeled nonfunction-blocking antibodies. We now demonstrate CALI of connexin43 (Cx43) and alpha1C L-type calcium channels, each tagged with one or two small tetracysteine (TC) motifs that specifically bind the membrane-permeant, red biarsenical dye, ReAsH. ReAsH-based CALI is genetically targeted, requires no antibodies or microinjection, and inactivates each protein by approximately 90% in <30 s of widefield illumination. Similar light doses applied to Cx43 or alpha1C tagged with green fluorescent protein (GFP) had negligible to slight effects with or without ReAsH exposure, showing the expected molecular specificity. ReAsH-mediated CALI acts largely via singlet oxygen because quenchers or enhancers of singlet oxygen respectively inhibit or enhance CALI.     

Generation of reactive oxygen species upon strong visible light irradiation of isolated phycobilisomes from Synechocystis PCC 6803

The mechanism of photodegradation of antenna system in cyanobacteria was investigated using spin trapping ESR spectroscopy, SDS-PAGE and HPLC-MS. Exposure of isolated intact phycobilisomes to illumination with strong white light (3500 micromol m(-2) s(-1) photosynthetically active radiation) gave rise to the formation of free radicals, which subsequently led to specific protein degradation as a consequence of reactive oxygen species-induced cleavage of the polypeptide backbone. The use of specific scavengers demonstrated an initial formation of both singlet oxygen (1O2) and superoxide (O2(-)), most likely after direct reaction of molecular oxygen with the triplet state of phycobiliproteins, generated from intersystem crossing of the excited singlet state. In a second phase carbon-based radicals, detected through the appearance of DMPO-R adducts, were produced either via O2(-) or by direct 1O2 attack on amino acid moieties. Thus photo-induced degradation of intact phycobilisomes in cyanobacteria occurs through a complex process with two independent routes leading to protein damage: one involving superoxide and the other singlet oxygen. This is in contrast to the mechanism found in plants, where damage to the light-harvesting complex proteins has been shown to be mediated entirely by 1O2 generation.     

Blood serum as a factor of chromatin condensation in trisomy-21 (Down's syndrome)]

The influence of blood sera from patients with Down's syndrome and healthy ones, and different serum fractions on the structure of the deoxyribonucleoprotein systems (DNP-system) has been studied. It was demonstrated that non-fractionated sera of the patients produce a condensation effect on the DNP-system in contradistinction to the sera from healthy people. The analysis of the action of single serum fractions showed that different condensation effect results from the activity of high molecular nondialysable thermosensitive components whose action disappears at gel-filtration of serum proteins. A possibility of the humoral control over chromatin structural organization in vivo is discussed in terms of the evidence on the similarity of serum proteins and chromosomal nonhistone proteins.     

Kinobeon A, purified from cultured safflower cells, is a novel and potent singlet oxygen quencher

We recently reported that kinobeon A, produced from safflower cells, suppressed the free radical-induced damage of cell and microsomal membranes. In the present study, we investigated whether kinobeon A quenches singlet oxygen, another important active oxygen species. Kinobeon A inhibited the singlet oxygen-induced oxidation of squalene. The second-order rate constant between singlet oxygen and kinobeon A was 1.15 x 10(10) M(-1)s(-1) in methanol containing 10% dimethyl sulfoxide at 37 degrees C. Those of alpha-tocopherol and beta-carotene, which are known potent singlet oxygen quenchers, were 4.45 x 10(8) M(-1)s(-1) and 1.26 x 10(10) M(-1)s(-1), respectively. When kinobeon A was incubated with a thermolytic singlet oxygen generator, its concentration decreased. However, this change was extremely small compared to the amount of singlet oxygen formed and the inhibitory effect of kinobeon A on squalene oxidation by singlet oxygen. In conclusion, kinobeon A was a strong singlet oxygen quencher. It reacted chemically with singlet oxygen, but it was physical quenching that was mainly responsible for the elimination of singlet oxygen by kinobeon A. Kinobeon A is expected to have a preventive effect on singlet oxygen-related diseases of the skin or eyes.     

Humoral antibodies to the antigens related to the structural protein antigens of the mammary cancer virus (MTV) in breast cancer patients and in healthy donors

Indirect radioimmunoassay and immunoperoxidase studies provided further evidence of human serum reactivity with murine mammary tumor virus (MMTV) structural proteins. Examination of over 400 human sera from breast cancer (BC) patients and controls has shown that the incidence of antibodies which react with MMTV structural proteins was significantly higher in BC patients (50%) than in patients with carcinomas of other organs (3%) or normal women (3%). However, the percentage of subjects immune to MMTV was higher in pregnant women (10%) as compared with normal subjects. All the women with BC of the first clinical stage had antibodies to MMTV. The percentage of immune donors was lower among patients with BC of the later stages (IIa, 90%, IIb, 76%, III, 27%, IV, 0%). Thus, a good agreement has been demonstrated between the first stages of BC and expression of antibodies to MMTV.     

Photosensitized protein damage by dimethoxyphosphorus(V) tetraphenylporphyrin

For the purpose of the basic study of photodynamic therapy, the activity of the water-soluble P(V)porphyrin, dimethoxyP(V)tetraphenylporphyrin chloride (DMP(V)TPP), on photosensitized protein damage was examined. The quantum yield of singlet oxygen generation by DMP(V)TPP (0.64) was comparable with that of typical porphyrin photosensitizers. Absorption spectrum measurement demonstrated the binding interaction between DMP(V)TPP and human serum albumin, a water-soluble protein. Photo-irradiated DMP(V)TPP damaged the amino acid residue of human serum albumin, resulting in the decrease of the fluorescence intensity from the tryptophan residue of human serum albumin. A singlet oxygen quencher, sodium azide, could not completely inhibit the damage of human serum albumin, suggesting that the electron transfer mechanism contributes to protein damage as does singlet oxygen generation. The decrease of the fluorescence lifetime of DMP(V)TPP by human serum albumin supported the electron transfer mechanism. The estimated contribution of the electron transfer mechanism is 0.64. These results suggest that the activity of DMP(V)TPP can be preserved under lower oxygen concentration condition such as tumor.     

Verteporfin, photofrin II, and merocyanine 540 as PDT photosensitizers against melanoma cells

The efficiency of photodynamic effect (PDE) for Photofrin II (PfII), Verteporfin, and Merocyanine 540 (MC540) was compared against neoplastic cells. Triplet state lifetimes and singlet molecular oxygen quantum yields were correlated with biological effect. PfII triplet lifetime was two times longer than that of Verteporfin, however, its singlet molecular oxygen quantum yield was two times lower in comparison with Verteporfin. High singlet molecular oxygen quantum yield of Verteporfin resulted in high biological efficacy. To achieve 50% mortality of cells four times lower light dose and five times lower concentration of Verteporfin were applied in comparison with PfII. The same level of cell damage was reached using 10 times higher light dose and two times higher concentration of MC540 in comparison with PfII. Our results confirm that singlet molecular oxygen based mechanism, prevalent for Verteporfin and PfII, was highly effective against melanoma cells. Verteporfin can be used at small doses with high cellular damage efficiency.     

In vitro phototoxicity of rhodopsin photobleaching products in the retinal pigment epithelium (RPE)

Although the primary biological function of retinal photoreceptors is to absorb light and provide visual information, extensive exposure to intense light could increase the risk of phototoxic reactions mediated by products of rhodopsin bleaching that might accumulate in photoreceptor outer segments (POS). The phototoxicity of POS, isolated from bovine retinas, was examined in cultured retinal pigment epithelium cells (ARPE-19) containing phagocytised POS and in selected model systems by determining POS ability to photogenerate singlet oxygen, and photoinduce oxidation of cholesterol and serum albumin. Bleaching of rhodopsin-rich POS with green light resulted in the formation of retinoid products exhibiting distinct absorption spectra in the near-UV. Irradiation of POS-fed ARPE-19 cells with blue light reduced their survival in a dose-dependent manner with the effect being stronger for cells containing prebleached POS. The specific and non-specific phagocytic activity of ARPE-19 cells was inhibited by sub-lethal photic stress mediated by phagocytised POS. The oxidising ability of POS photobleaching products was demonstrated both in a model system consisting of serum albumin and in ARPE-19 cells. Distinct photooxidation of proteins, mediated by POS, was observed using coumarin boronic acid as a sensitive probe of protein hydroperoxides. Irradiation of POS with blue light also induced oxidation of liposomal cholesterol as determined by HPLC-EC(Hg). Time-resolved singlet oxygen phosphorescence demonstrated the efficiency of retinoids, extracted from POS by chloroform-methanol treatment, to photogenerate singlet oxygen. The results indicate that photic stress mediated by POS photobleaching products could inhibit phagocytic efficiency of RPE cells and, ultimately, compromise their important biological functions.     

Mechanism of DNA cleavage induced by sodium chromate(VI) in the presence of hydrogen peroxide

Reactivities of chromium compounds with DNA were investigated by the DNA sequencing technique using 32P 5'-end-labeled DNA fragments, and the reaction mechanism was investigated by ESR spectroscopy. Incubation of double-stranded DNA with sodium chromate(VI) plus hydrogen peroxide or potassium tetraperoxochromate(V) led to the cleavage at the position of every base, particularly of guanine. Even without piperidine, the formation of oligonucleotides was observed, suggesting the breakage of the deoxyribose-phosphate backbone. ESR studies using hydroxyl radical traps demonstrated that hydroxyl radical is generated both during the reaction of sodium chromate(VI) with hydrogen peroxide and the decomposition of potassium tetraperoxochromate(V), and that hydroxyl radical reacts significantly not only with mononucleotides but also with deoxyribose 5-phosphate. ESR studies using a singlet oxygen trap demonstrated that singlet oxygen is also generated both by the same reaction and decomposition, and reacts significantly with deoxyguanylate, but scarcely reacts with other mononucleotides. Furthermore, ESR studies suggested that tetraperoxochromate(V) is formed by the reaction of sodium chromate(VI) with hydrogen peroxide. These results indicate that sodium chromate(VI) reacts with hydrogen peroxide to form tetraperoxochromate(V), leading to the production of the hydroxyl radical, which causes every base alteration and deoxyribose-phosphate backbone breakage. In addition, sodium chromate(VI) plus hydrogen peroxide generates singlet oxygen, which subsequently oxidizes the guanine residue. The mechanism by which both hydroxyl radical and singlet oxygen are generated during the reaction of sodium chromate(VI) with hydrogen peroxide was presented. Finally, the possibility that this reaction may be one of the primary reactions of carcinogenesis induced by chromate(VI) is discussed.     

Protein oxidation and proteolysis by the nonradical oxidants singlet oxygen or peroxynitrite

Exposure of proteins to oxidants leads to increased oxidation followed by preferential degradation by the proteasomal system. The role of the biologically occurring oxidants singlet oxygen and peroxynitrite in oxidation of proteins in living cells and enhanced degradation of these proteins was examined in this study. Subsequent to treatment of an isolated model protein, ferritin, with singlet oxygen or peroxynitrite, there was enhanced degradation by the isolated 20S proteasome. Treatment of clone 9 liver cells (normal liver epithelia) with two different singlet oxygen-generating systems or peroxynitrite leads to a concentration-dependent increase in cellular protein turnover. At high concentrations of these oxidants, the protein turnover decreases without significant loss of cell viability and proteasome activity. To compare the increase of intracellular protein turnover with that obtained with other oxidants, cells were exposed to hydrogen peroxide or xanthine/xanthine oxidase. The maximal increase in protein turnover was similar with the various oxidants. The oxidized protein moieties were removed by enhanced protein turnover. Removal of singlet oxygen- or peroxynitrite-damaged proteins is dependent on the proteasomal system, as suggested by the sensitivity to lactacystin. Our results provide evidence that the proteasomal system is able to selectively recognize and degrade proteins modified by singlet oxygen or peroxynitrite in vitro as well as in living cells.     

Rates of pinocytic capture of simple proteins by rat yolk sacs incubated in vitro.

The rates of pinocytic uptake of a number of small 125I-labelled simple proteins (insulin, ribonuclease A and lysozyme) by rat yolk sacs incubated in vitro were determined both before and after treating these proteins with reagents that are known to increase the rate of capture of 125I-labelled bovine serum albumin. Uptake of the untreated forms of all three proteins was extremely rapid, indicating that adsorptive pinocytosis is the principal mechanism by which yolk-sac cells capture these simple proteins, but these rates show no simple correlation with molecular charge. In contrast with albumin, the rates of uptake of treated proteins were either unchanged or lower than that of the corresponding untreated protein preparations; polymeric forms of 125I-labelled lysozyme larger than dimers were ingested at rates significantly lower than that of the monomer.     

Photo-excitation of carotenoids causes cytotoxicity via singlet oxygen production

Carotenoids, natural pigments widely distributed in algae and plants, have a conjugated double bond system. Their excitation energies are correlated with conjugation length. We hypothesized that carotenoids whose energy states are above the singlet excited state of oxygen (singlet oxygen) would possess photosensitizing properties. Here, we demonstrated that human skin melanoma (A375) cells are damaged through the photo-excitation of several carotenoids (neoxanthin, fucoxanthin and siphonaxanthin). In contrast, photo-excitation of carotenoids that possess energy states below that of singlet oxygen, such as β-carotene, lutein, loroxanthin and violaxanthin, did not enhance cell death. Production of reactive oxygen species (ROS) by photo-excited fucoxanthin or neoxanthin was confirmed using a reporter assay for ROS production with HeLa Hyper cells, which express a fluorescent indicator protein for intracellular ROS. Fucoxanthin and neoxanthin also showed high cellular penetration and retention. Electron spin resonance spectra using 2,2,6,6-tetramethil-4-piperidone as a singlet oxygen trapping agent demonstrated that singlet oxygen was produced via energy transfer from photo-excited fucoxanthin to oxygen molecules. These results suggest that carotenoids such as fucoxanthin, which are capable of singlet oxygen production through photo-excitation and show good penetration and retention in target cells, are useful as photosensitizers in photodynamic therapy for skin disease.     

Immune response in subacute sclerosing panencephalitis: reduced antibody response to the matrix protein of measles virus.

Immune precipitation was used to study the humoral immune response of patients with subacute sclerosing panencephalitis (SSPE). Patients with SSPE have a progressive infection of the CNS by measles or a measles variant despite high serum antibody levels to measles virus as measured by standard serologic techniques. However, when the antibody response to individual measles virus proteins was measured, we found a striking reduction in the ability of sera from patients with SSPE to precipitate the matrix (M) protein as compared to the precipitation of the M protein by sera from normal adults who had natural measles infection in childhood, or by convalescent sera obtained 3 to 5 weeks after a naturally occurring measles infection. The decreased antibody response to the M protein in sera from patients with SSPE occurred despite a vigorous antibody response to the other viral proteins, suggesting a selective defect in the production of antibody to a single viral protein. The reduced anti-M antibody in sera from patients with SSPE was demonstrated whether immune precipitation was performed with wild-type measles virus or SSPE virus proteins. These results suggest that in SSPE only small amounts of the M protein are produced. This result may help explain how measles virus persists in the central nervous system of patients with SSPE.     

Yeast thioredoxin peroxidase expression enhances the resistance of Escherichia coli to oxidative stress induced by singlet oxygen

Singlet oxygen ((1)O(2)) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. A soluble protein from Saccharomyces cerevisiae specifically provides protection against a thiol-containing metal-catalyzed oxidation system (thiol/Fe(3+)/O(2)) but not against an oxidation system without thiol. This 25 kDa protein acts as a peroxidase but requires the NADPH-dependent thioredoxin system or a thiol-containing intermediate, and was named thioredoxin peroxidase (TPx). The role of TPx in the cellular defense against oxidative stress induced by singlet oxygen was investigated in Escherichia coli containing an expression vector with a yeast genomic DNA fragment that encodes TPx and mutant in which the catalytically essential amino acid cysteine (Cys-47) has been replaced with alanine by a site-directed mutagenesis. Upon exposure to methylene blue and visible light, which generates singlet oxygen, there was a distinct difference between the two strains in regard to growth kinetics, viability, the accumulation of oxidized proteins and lipids, and modulation of activities of superoxide dismutase and catalase. The results suggest that TPx may play an important protective role in a singlet oxygen-mediated cellular damage.     

Effect of ascorbic acid on the production of singlet oxygen by purified human myeloperoxidase

We have previously studied purified human myeloperoxidase-hydrogen peroxide-halide ion systems as models of possible singlet oxygen production by granulocytes. While myeloperoxidase could efficiently produce singlet oxygen, the yield of singlet oxygen at a physiological pH with Cl- was very small due to enzyme inactivation. In that Bolscher et al. [(1984) Biochim. Biophys. Acta 784, 189-191] observed that micromolar concentrations of ascorbic acid prevented inactivation of myeloperoxidase and increased the production of hypochlorous acid, we examined whether ascorbic acid would augment singlet oxygen production by the myeloperoxidase-hydrogen peroxide-halide ion systems. Ascorbic acid, however, fails to increase the singlet oxygen yield, suggesting that it does not augment singlet oxygen production in the intact granulocyte by a myeloperoxidase-dependent mechanism.