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J Everse

Publications and source records attributed to J Everse.

At least 19 recordsLinked to original sources
PubMed1998

The structure of heme proteins Compounds I and II: some misconceptions.

Many reactions catalyzed by heme proteins involve an oxidation of the heme to one or two equivalents above the ferric state. Such intermediates are often referred to as Compound II and Compound I, respectively. Several different notations are used in the literature to describe the chemical structures of these compounds, which has led to errors and misinterpretations. The main problems are: 1. For many biochemists the notations X - FeIV = O and X - Fe + = O are equivalent and are used interchangeably, whereas other biochemists interpret these notations to have quite different meanings. 2. It is inaccurate and misleading to illustrate the increased oxidation state of Compound I by just adding two positive charges to the structure. 3. The bond between the oxygen and iron is not a conventional double bond and illustrating it as such leads to misconceptions concerning its properties. 4. In several instances, including horseradish peroxidase Compound I, there is reason to doubt that the radical moiety of the porphyrin ring (or of the protein in other peroxidases) carries a positive charge. The purpose of this article is to promote the use of uniform, as well as chemically correct, formulae and equations in describing the structures and reactivities of Compounds I and II. To accomplish this, a new notation is proposed for the iron-oxygen bond in these compounds.

Animals↗
PubMed1998

Synergism of dimethoxybenzosemiquinone free radicals and CD4+ T-lymphocytes to suppress Ehrlich ascites tumor.

Numerous natural and synthetic quinone compounds possess significant antitumor properties. Various mechanisms have been proposed to account for these properties, including scission and degradation of tumor cell DNA, intracellular "redox cycling" to cogenerate semiquinone free radicals and reactive oxygen intermediates, and the interaction of semiquinone radicals with tumor cell surface flavoenzymes. However, no evidence has been presented to explain adequately the preferential attack on tumor cells by semiquinone radicals, as opposed to normal cells. To address this question, a synergistic interaction was examined. A therapy consisting of a mixture of L-ascorbate and 2,6-dimethoxy-p-benzoquinone, was used for in vivo evaluation. BALB/c mice were depleted of functional T-lymphocytes by xenogeneic monoclonal antibody pretreatment, challenged with Ehrlich ascites tumor, and administered the semiquinone radical-generating therapy. Mice depleted of CD4+ T-lymphocytes responded with rapidly fatal tumor progression, with significantly decreased mean survival times over controls, whereas less severe responses were observed in mice devoid of CD8+ T-lymphocytes. Mice depleted of both T-lymphocyte subpopulations responded with uninhibited tumor growth and rapid mortalities. When tumor challenge occurred after therapy, tumor growth was significantly delayed in mice enriched for CD4+ T-lymphocytes, with demonstrable increases in mean survival time over controls. This reagent combination had no significant effect on T-lymphocyte profiles in secondary lymphoid organs. These data suggest a synergistic phenomenon of semiquinone radical-induced cytostasis coupled with T-lymphocyte helper activity for optimal tumor suppression.

Animals↗
PubMed1997

The toxicities of native and modified hemoglobins.

Recent research on the potential use of hemoglobin derivatives as a blood substitute has revealed that the administration of large quantities of free hemoglobin into the circulation results in a variety of toxic side effects. Because it has been well established that hemoglobin, like myoglobin, has considerable pro-oxidant activity, a number of studies have appeared suggesting that the administered hemoglobins may catalyze various oxidative and peroxidative reactions, which in turn, would cause the observed pathologic conditions. This occurs as a result of the in vivo formation of highly oxidized forms of the native and modified hemoglobins. In addition, it has been proposed that considerable amounts of free hemin and iron may be generated as a result of the catabolism of the injected hemoglobin. Hemin is known to be very toxic when present in large amounts, and iron could catalyze the formation of hydroxyl radicals via Fenton-type reactions. Thus, the toxic activities of catabolic products of hemoglobin could also be involved in all or part of the observed side effects. The purpose of this review is to consider the conditions under which reactive species of hemoglobin may be formed in vivo, their potential reactivity, and whether their individual or combined oxidative activities could account for the biological damage that is observed in vivo following hemoglobin transfusions.

Blood Substitutes↗
PubMed1996

The cytotoxic activities of human hemoglobin and diaspirin crosslinked hemoglobin.

It is well known that hemoglobin (Hb) possesses many oxidative enzyme activities, including a pseudo-peroxidase activity. It has also been shown by many investigators that various peroxidases in the presence of hydrogen peroxide and a halide ion exert a potent cytotoxic activity toward various mammalian cell types. It has further been observed by various investigators that the administration of relatively large amounts of purified Hb or a Hb derivative to a host animal during resuscitation experiments leads to a number of unrelated types of tissue damage and cell damage in the host. The first objective of this investigation was to determine if the observed tissue and cell damage may be due to a cytotoxic activity that Hb may exert in vivo analogous to that of the peroxidases. We also showed some time ago that peroxidases are able to activate peritoneal macrophages to the cytocidal state. Hence, we also addressed the question whether or not Hb is able to activate macrophages in a similar manner. Our results were negative with regard to both questions. Further investigations indicated that, unlike the peroxidases, ferryl-Hb is unable to oxidize iodide to iodine at a measurable rate, which appears to be the reason for the lack of cytotoxic activity.

Animals↗
PubMed1995

Probable immune system mediation of the antitumor activity of the 2,6-dimethoxybenzo-p-semiquinone radical.

We recently reported that the antitumor activity of an immobilized oxidase-peroxidase system, which can produce radical species as intermediates, is dependent on the presence of an intact and functioning immune system in the host. A number of recent investigations have indicated that the bioactive form of many quinone-type anticancer agents may be the semiquinone-type radical. To investigate if the antitumor activity of some of the quinone-type anticancer agents may also be dependent on a fully functioning immune system in the host, we used as a simple, well-defined model the 2,6-dimethoxybenzo-p-semiquinone radical. This free radical has a potent antitumor activity against Ehrlich ascites tumors in mice, as first described by Szent-Gyorgyi's group. In the present study, we investigated its antitumor activity in immunocompromised BALB/c mice. We found that the antitumor activity of the semiquinone radical is completely abolished in mice that had previously been subjected to whole body gamma irradiation, indicating a possible involvement of one or more cells of the host's defense system. The antitumor activity was not abolished in mice with dysfunctional macrophages induced by a severe selenium deficiency. However, the radical was inactive in mice whose immune system was suppressed with cyclosporin A and in nude (nu/nu) mice. These results suggest that the dimethoxybenzo-semiquinone radical, rather than acting primarily as a cytotoxic agent as previously reported, exerts its antitumor activity mainly via a more complex mechanism in which T lymphocytes appear to play a major role.

Animals↗
PubMed1993

Antitumor activity of an immobilized peroxidase system against murine Ehrlich ascites is mediated by the immune system.

Earlier studies have shown that a mixture of glucose oxidase and a peroxidase exerts a tumoricidal effect on rats bearing Novikoff hepatomas when the enzyme mixture is injected intraperitoneally. The enzyme mixture was shown to be nontoxic when injected into healthy animals at levels up to 600 times the therapeutic dose. In the present study, we have evaluated the possibility that the host immune defense system may be involved in the antitumor activity of the peroxidase system, using the murine Ehrlich ascites tumor as the target. The results revealed that the antitumor activity of the peroxidase system is absent in tumor-bearing animals whose immune system has been compromised by whole body gamma-irradiation or by an induced selenium deficiency. The peroxidase system was also found to be inactive in tumor-bearing mice whose immune system was suppressed by the administration of cyclosporin A as well as in athymic (nu/nu) mice. These results indicate that T lymphocytes may directly or indirectly be involved in the in vivo antitumor activity of the peroxidase system. This could explain the observed high selectivity toward tumor cells by the enzyme system in vivo and its lack of toxicity in healthy animals.

Animals↗
PubMed1992

Pilot scale production of pyrogen-free human hemoglobin for research.

A pilot scale production facility for the preparation of 20 to 30 liters of stroma-free hemoglobin is described. The system is capable of producing pyrogen-free solutions for research purposes. It is not certified for production of parenteral solutions for human use, but the plan could be implemented to meet standards for such materials. Products of the facility should be of adequate quality to address most of the toxicity and efficacy issues facing further development of hemoglobin-based red cell substitutes.

Biotechnology↗
PubMed1988

Peroxidase-induced enhancement of chemiluminescence by murine peritoneal macrophages.

A number of substances have been shown to enhance the respiratory burst (RB) of macrophages. Many of these substances are not normally found in vivo. The present study suggests that a group of enzymes characterized as peroxidases have the ability to significantly enhance the RB and concomitant phagocytosis by murine peritoneal macrophages. Horseradish peroxidase (HRP), lactoperoxidase (LPO), and microperoxidase (MPO) can significantly augment these functions. Both resident and thioglycollate-induced macrophages exhibited enhanced chemiluminescence (CL) upon exposure to HRP, however, the effect was more pronounced with the latter. The increase in CL was correlated with an increase in production of superoxide, which was measured by reduction of cytochrome c. Horseradish peroxidase immobilized on an inert carrier, was capable of enhancing the RB suggesting that it does not have to enter the cell in order to function. Hemin, hematoheme and hematoporphyrin had little effect on macrophage stimulated CL. All of the peroxidases tested caused increased phagocytosis of opsonized zymosan. These studies indicate that peroxidases are capable of stimulating the RB, phagocytosis and possibly other macrophage functions.

Animals↗
PubMed1988

Endotoxemia and neutrophil activation in vivo.

There is a growing body of data to suggest that marginated granulocytes mediate much of the pulmonary damage observed during endotoxemia. The mechanism(s) by which endotoxemia initiates neutrophil margination and cytotoxicity remain either controversial or unknown. The objectives of this study were 1) to determine the temporal relationship between endotoxin-induced decreases in mean arterial pressure and circulating neutrophils, 2) to monitor neutrophil activation in vivo by measuring myeloperoxidase (MPO) activity in the plasma and lymph, and 3) to assess the interaction between endotoxin and complement in activation of neutrophilic oxidative metabolism in vitro. We found that a bolus injection of endotoxin causes a concurrent decrease in both mean arterial pressure and circulating neutrophils at 2 min postinfusion. Blood pressure recovered to approximately 70% of control values by 180 min, whereas circulating neutrophils remain depressed at 20% of control values for the entire experimental period. Using MPO as a marker for neutrophil activation, we found that infusion of endotoxin produces a dramatic increase in plasma and lymph MPO activity, suggesting activation of neutrophilic metabolism in vivo. In vitro data showed that both endotoxin and plasma were required for optimal neutrophilic degranulation and superoxide formation. We conclude that 1) the appearance of MPO in the plasma (or lymph) may be a useful neutrophil marker for neutrophil activation in vivo and may prove useful in following the course of neutrophil-mediated tissue injury during endotoxemia, and 2) endotoxin-activated complement (C5a) activates neutrophils to produce cytotoxic oxidants.

Animals↗
PubMed1988

The mechanism of peroxidase-mediated cytotoxicity. II. Role of the heme moiety.

Various peroxidases in the presence of hydrogen peroxide and a halide ion have been shown to exert a cytolytic activity against erythrocytes and other cells. However, few studies have been done to elucidate the active site on the enzymes that is responsible for the cytotoxic activity. In addressing this question we found that boiling of horseradish peroxidase only partially abolishes its cytotoxic activity, suggesting that an intact tertiary structure of the protein may not be essential for the cytotoxic activity. This conclusion was confirmed by demonstrating that microperoxidase, hemin, and hematoheme also exert cytotoxic activity in the presence of hydrogen peroxide and iodide, the kinetics of which were identical to those obtained with the peroxidases. Fluoride, bromide, and thiocyanate could not replace iodide in any of these systems. These results indicate that the active site for the cytotoxic activity of the peroxidases is located within the heme moiety, whereas the protein portions of the enzymes affect the cytotoxic activity of the enzymes only in an indirect manner. We also tested a variety of compounds for their ability to inhibit the cytolytic reaction toward erythrocytes. We found that compounds such as thiourea, thionicotinamide, and uric acid are much more potent inhibitors of the cytolytic reaction than tyrosine and histidine. These observations support the concept that oxidative reactions rather than halogenation reactions are the primary cause of the peroxidase-mediated lysis of erythrocytes.

Animals↗
PubMed1987

The cytotoxic activity of hematoheme: evidence for two different mechanisms.

Hematoheme displays a potent cytolytic activity toward erythrocytes either in the presence of hydrogen peroxide and a halide ion (system I) or in the presence of oxygen and a reducing agent (system II). In system I it resembles the cytotoxic activity of various peroxidases, whereas in system II it resembles the destructive activity of bleomycin and a variety of metal complexes. Both types of reactions presumably involve the generation of active oxygen species, which are responsible for the damaging effects. In a first attempt to compare the chemical mechanisms of the two types of reactions we used various traps and scavengers of active oxygen species. Tryptophan as well as tyrosine and uric acid were found to be potent inhibitors of the hematoheme-H2O2-halide reaction but do not significantly inhibit the hematoheme-O2-ascorbate reaction. Pyridine, on the other hand, inhibits the oxygen-mediated reaction, but does not affect the peroxide-halide-mediated activity. The cytolytic activity of photoactivated hematoporphyrin, which involves the generation of singlet oxygen, is activated by pyridine and is strongly inhibited by diphenylisobenzofuran. The latter compound is a weak activator of both hematoheme reactions. We conclude that the two hematoheme reactions proceed by two different mechanisms and probably generate different toxic intermediates. The results further suggest that the toxic intermediate generated by photoactivated hematoporphyrin (singlet oxygen) does not play a dominant role in either of the two hematoheme reactions.

Animals↗
PubMed1987

Neuromelanogenic and cytotoxic properties of canine brainstem peroxidase.

We have isolated a heme protein from canine midbrains that possesses potent peroxidase activity. This enzyme catalyzes the oxidation of dopamine to neuromelanin in the presence of H2O2. We have further shown that the isolated peroxidase possesses potent cytotoxic activity in the presence of superoxide or H2O2 and Cl-. The enzyme possesses an endogenous NAD(P)H oxidase activity that can promote the cytotoxic activity by virtue of its production of superoxide. Other enzymes such as dihydroorotate dehydrogenase and galactose oxidase, which produce O2- and H2O2, respectively, are also effective in promoting the cytotoxic activity of the brainstem peroxidase. Although rat erythrocytes were routinely used as the target cell, other cell types, including rat hepatoma and mouse neuroblastoma cells, are also susceptible to the toxic action of the peroxidase. The cytotoxic action of the brainstem peroxidase is dramatically enhanced by kainic acid and is significantly enhanced by Mn2+, whereas dopamine was found to be a potent inhibitor of the cytotoxic activity. Based on these findings, we postulate a central role for the brainstem peroxidase in dopamine metabolism as well as in the biochemical and anatomical changes associated with Parkinson's disease.

Animals↗
PubMed1986

The cytotoxic activity of hematoporphyrin: studies on the possible role of transition metals.

Hematoporphyrin acquires a potent cytolytic activity toward erythrocytes when activated by visible light. Considerable evidence has been obtained suggesting that this toxic activity is mediated by certain active oxygen species, including singlet oxygen and hydroxyl radicals. These active oxygen species have also been proposed as intermediates in the toxic activity of peroxidases, hemin, and a variety of metal complexes. Unlike hematoporphyrin, all these compounds contain a liganded Fe atom, which appears to play a central role in the activation of molecular oxygen. In order to ascertain whether the generation of active oxygen by hematoporphyrin may also involve the participation of a metal ion we have compared the cytolytic activity of hematoporphyrin with that of hematoheme. The participation of a metal ion in the light-activated hematoporphyrin reaction was ruled out on the basis of four criteria: no increase in cytolytic activity was observed upon the addition of Fe or Cu ions; no evidence could be obtained for the incorporation of a metal ion into hematoporphyrin during light activation; hematoporphyrin is a more potent cytolytic agent than hematoheme on an equimolar basis; and the activities of the two cytolytic agents are affected differently by various activators and inhibitors of the toxic reaction. Our results further indicate that the mechanism of the cytolytic activity promoted by light-activated hematoporphyrin is distinctly different from that promoted by hematoheme in the presence of ascorbate. We conclude that the two cytolytic reactions are most likely propagated by two different forms of active oxygen.

Animals↗
PubMed1986

Activation of macrophages by peroxidases.

Peritoneal macrophages from C57BL/6 mice were activated in vitro with various peroxidases and their cytotoxic activity toward 3T12 cells was determined. Destruction of 3T12 cells by macrophages stimulated with horseradish peroxidase, lactoperoxidase, and microperoxidase was observed at peroxidase concentrations as low as 9, 1.6, and 200 nM, respectively. A 50% cytotoxic effect was obtained at peroxidase concentrations of 0.9, 1.6, and 1.5 microM, respectively. The macrophage-stimulating activity of horseradish peroxidase was not destroyed by boiling. This, together with the high activity of microperoxidase, indicates that the macrophage-stimulating activity of the peroxidases is probably associated with the heme portion of the enzymes. On a molar basis the peroxidases are much less potent macrophage activators than interferon (alpha + beta) and endotoxin. Nevertheless, our data clearly indicate that peroxidases are a group of enzymes capable of inducing macrophage activation, resulting in cytostatic and/or cytocidal activity.

Animals↗