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The content of nitric oxide in convulsions of different genesis is assessed by measuring the formation of paramagnetic mononitrosyl iron complexes with diethylthiocarbamate by electron paramagnetic resonance. A 3- to 4-fold increase in the content of these complexes is found in the brain of rats with thiosemicarbazide- or N-methyl-DL-aspartate-induced seizures in comparison with control animals. A similar increase in the brain NO content was observed in maximum electrical stimulation of metrazol-induced convulsions. These changes were accompanied by elevation of secondary lipid peroxidation products.

We have examined the effect of the dinitrosyl iron complexes, the NO donors, on the resistance of isolated heart to ischemia and reperfusion, as well as the effect of quercetin, an inhibitor of HSP70 transcription, on NO-induced enhancement of cardiac resistance to ischemia and reperfusion. The iron complexes were found to produce dose-and time-dependent protective effect and enhance the resistance of isolated heart to rhythm and contraction disorders during reperfusion. The maximum protective effect was observed 12–24 h postinjection of the 200 mg/kg dose. Quercetin completely prevented protective effect of the NO donor. The property of NO donors to stimulate HSP70 synthesis and the present data indicate that NO-dependent activation of HSP70 synthesis is a natural mechanism of cardiac protection against disturbances provoked by ischemia and reperfusion.

Parameters of the EPR signals of monomeric dinitrosyl-iron complexes with 1H-1,2,4-triazole-3-thiol (DNIC-MT), obtained by treating MT+ferrous iron in DMSO solution with gaseous NO, have been compared with those of the crystalline monomeric DNIC-MT with tetrahedral structure. Dissolved DNIC-MT were characterized by the isotropic EPR signal centered at g=2.03 with half-width of 0.7 mT and quintet hyperfine structure when recorded at ambient temperature or the anisotropic EPR signal with g( perpendicular)=2.045, g( parallel)=2.014 from frozen solution at 77 kappa, Cyrillic. DNIC-MT in crystalline state showed the structure-less symmetrical singlet EPR signal centered at g=2.03 and half-width of 1.7 mT at both room and liquid nitrogen temperature. The Lorentz shape of this signal indicates the strong exchange interaction between these complexes in the DNIC-MT crystal. Being dissolved in DMSO the crystalline sample of DNIC-MT demonstrated the EPR signal typical for DNIC-MT, obtained by treating MT+ferrous iron in DMSO solution with gaseous NO. Low spin (S=1/2) d(9) electron configuration of DNIC-MT with tetrahedral structure (formula [(MT-S(.))(2)Fe(-1)(NO(+))(2)](+)) was suggested to be responsible for the signal of DNIC-MT in crystalline state. Dissolving of the crystals of DNIC-MT may result in the change of their spatial and electronic structure, namely, tetrahedral structure of the complexes characterized by low spin d(9) electronic configuration transforms into a plane-square structure with d(7) electronic configuration and low spin S=1/2 state (formula [(MT- S(-))(2)Fe(+)(NO(+))(2)](+)). The latter was suggested to be characteristic of other DNICs with various thiol-containing ligands in the solutions. The proposed mechanism of these DNICs formation from ferrous iron, thiol and NO shows that the process could be accompanied by the ionization of NO molecules to NO(+) and NO(-) ions in the complexes. Detailed analysis of the shape of the EPR signals of these complexes provided additional information about the exchange interaction typical for DNIC-MT in crystals.

As our group has shown, the NO-synthase inhibitor L-NNA decreased 2–3 times heat shock-induced synthesis of the heat shock protein HSP70 (FEBS Lett. 370 (1995) 159–162). It was suggested that NO is involved in such induction. In the present study, it was found that (1) injection of the NO donor dinitrosyl iron complex (DNIC) into rats results in accumulation of HSP70 in the heart; (2) heat shock is accompanied by increased generation of NO (EPR assay) and HSP70 accumulation in cultured cells; (3) DNIC induces HSP70 accumulation in cultured cells not exposed to heat shock.

The effect of CCl4 on the generation of NO in mouse liver cells is studiedin vivo. Injection of CCl4 is shown to modulate the synthesis of NO by activating the NO-synthetase system. The experimental data suggest that O2− plays an essential role in the regulation of NO-synthetase system.

Intermittent daily exposures (60 s) to NO-containing gas flow (NO dose of 500 ppm) generated by air-plasma unit "Plason" improves healing of skin wounds in rats. The gas flow treatment shortened the recovery time of both aseptic and purulent wounds (300 mm2 area) by nearly a third. The treatment allows to achieve a marked improvement in the histological, histochemical, and electron-microscopic characteristics of the affected tissue. The mechanism of this phenomenon was studied by spin trapping method. The NO status of the wound tissue was investigated with EPR by following the formation of paramagnetic mononitrosyl complexes with iron-diethyldithiocarbamate, or with the heme groups in hemoglobin or myoglobin. For the first 5 min after a gas treatment with the exposure of 60s, detectable NO levels in the affected tissue were slightly lowered with respect to untreated controls. At subsequent times, treated tissues showed the formation of large quantities of nitroso-iron complexes: At 30-40 min after gas exposure, their levels were nearly two orders of magnitude higher than soon after (15 s-5 min) the exposure. The data demonstrate that the accumulation of nitrosyl-iron complexes reflects a sharp rise in endogenous NO production inside the affected tissue. Paradoxically, the beneficial effect of gaseous NO treatment can be mediated by the formation of limited quantities of peroxynitrite due to the reaction between exogenous NO and superoxide anions generated in high amount in wound tissue. This peroxynitrite has a strong prooxidant effect and can activate various antioxidant systems which diminish the amount of superoxide anions in wound tissue. The reduced superoxide levels allow to increase the contents of endogenous NO in gas-treated tissues. Therefore, the beneficial action of the treatment is attributed to enhanced NO bioavailability.

Nitrosyl-iron complexes used as aqueous preparations of binuclear dinitrosyl-iron complex with glutathione (DNICglu), initially polycrystalline preparations of binuclear tetranitrosyl-iron complex with thiosulfate (TNICthio), and also binuclear tetranitrosyl-iron complex with aminotriazole (TNICatria) and mononuclear dinitrosyl-iron complex with triazole (DNICtria) in the concentration to 0.1 mM activated expression of the soxS and sfiA genes in Escherichia coli. Higher concentrations of polycrystalline preparations of low stability in aqueous solutions were cytotoxic, whereas DNICglu, which is more stable in water (up to two days), increased the gene expression on increase in its concentration to 0.5 mM. The iron chelating agent o-phenanthroline completely inhibited the gene expression induced by all compounds studied. The genetic signal transduction seemed to be realized not by nitric oxide molecules and/or iron ions released in solutions but directly by the complexes themselves, which activate transcriptional proteins by transfer onto them of nitrosyl-iron groups [Fe+(NO+)2].

The inhibition of arterial tone produced by two nitric oxide (NO) derivatives of biological relevance, dinitrosyl–iron complexes with cysteine (DNIC-CYS) or with glutathione (DNIC-GSH), was compared. Both compounds induced vasorelaxation within the same concentration range (3–300 nM) in endothelium-denuded rat aortic rings. Consistent with a faster rate of NO release from DNIC-CYS than from DNIC-GSH, the relaxant effect of DNIC-CYS was rapid in onset and tended to recover with time, whereas the one of DNIC-GSH developed slowly and was sustained. In addition, DNIC-GSH (0.3 and 1 μM) but not DNIC-CYS (1 μM) induced, even after washout of the drug, a persistent hyporesponsiveness to vasoconstrictors and a relaxant effect of low molecular weight thiols like N -acetylcysteine (NAC, which can displace NO from preformed NO stores). Both effects of DNIC-GSH were associated with elevation of cyclic GMP content and were attenuated by NO scavengers or a cyclic GMP-dependent protein kinases inhibitor. In rings previously exposed to DNIC-GSH, addition of mercuric chloride (which can cleave the cysteineNO bond of S -nitrosothiols) elicited relaxation, completely blunted the one of NAC and also abolished the persistent elevation of NO content. In conclusion, this study shows that whereas both DNIC-CYS and DNIC-GSH elicited a NO release-associated relaxant effect in isolated arteries, only DNIC-GSH induced an inhibition of contraction which persisted after drug removal. The persistent effect of DNIC-GSH was attributed to the formation of releasable NO stores in arterial tissue, most probably as S -nitrosothiols. Thus, the nature of the thiol ligand plays a critical role in determining the mechanisms and duration of the effect of LMW-DNIC in arteries.

We studied the capability of dimeric forms of dinitrosyl-iron complexes and S-nitrosothiols to activate soluble guanylate cyclase (sGC) from human platelet cytosol. The dinitrosyl-iron complexes had the ligands glutathione (DNIC-GS) or N-acetylcysteine (DNIC-NAC). The S-nitrosothiols were S-nitrosoglutathione (GS-NO) or S-nitrosoacetylcysteine (SNAC). For both glutathione and N-acetylcysteine, the DNIC and S-nitrosothiol forms are equally effective activators of sGC. The activation mechanism is strongly affected by the presence of intrinsic metal ions. Pretreatment with the potent iron chelator, disodium salt of bathophenanthroline disulfonic acid (BPDS), suppressed sGC activation by GS-NO: the concentration of GS-NO producing maximal sGC activation was increased by two orders of magnitude. In contrast, activation by DNIC-GS is strongly enhanced by BPDS. When BPDS was added 10 min after supplementation of DNIC-GS or GS-NO at 4 degrees C, it exerted a similar effect on sGC activation by either NO donor: BPDS only enhanced the sGC stimulation at low concentrations of the NO donors. Our experiments demonstrated that both Fe(2+) and Cu(2+) ions contribute to the decomposition of GS-NO in the presence of ascorbate. The decomposition of GS-NO induced by Fe(2+) ions was accompanied by formation of DNIC. BPDS protected GS-NO against the destructive action of Fe(2+) but not Cu(2+) ions. Additionally, BPDS is a sufficiently strong chelator to remove the iron from DNIC-GS complexes. Based on our data, we propose that S-nitrosothiols activate sGC via a two-step iron-mediated process: In the first step, intrinsic Fe(2+) ions catalyze the formation of DNICs from S-nitrosothiols. In the secondary step, these newly formed DNICs act as the real NO donors responsible for sGC activation.

Nitric oxide (NO) generation in the brain cortex of Wistar rats was measured by direct method of electron paramagnetic resonance (EPR) spectroscopy. Dramatic (fivefold) elevation of NO production was found during penthylenetetrazole (PTZ)-induced epileptiform seizures. The level of secondary products of lipid peroxidation (LPO; thiobarbituric acid reactive substances, TBARS) was also significantly increased in the cerebral cortex of rats with PTZ-evoked seizures. The effects of anticonvulsant drugs phenobarbital, lamotrigine, phenazepam, as well as antioxidant substances alpha-tocopherol and novel original Russian synthetic drug mexidol (2-ethyl-6-methyl-3-oxypiridine succinate), were investigated. All the substances studied significantly decreased seizure manifestations and partially prevented both enhancement of NO generation and increase in TBARS formation. Mexidol and phenobarbital were found to be the most effective in the preventing of PTZ-induced seizures among all the substances studied. The data obtained support our speculation that neuroprotective action of mexidol may correlate with its ability to inhibit not only excessive reactive oxygen species (ROS) formation but also NO generation. While the molecular mechanism underlying action of mexidol and phenobarbital still remains unclear, it is likely that the effect of these drugs on NO production is contributing to their neuroprotective action. It might be concluded that both the suppression of seizure-induced NO generation and LPO enhancement may be involved in the mechanism of action of antiepileptic drugs.

The present experiments were designed to analyze the influence of copper and iron ions on the process of decomposition of S-nitrosocysteine (cysNO), the most labile species among S-nitrosothiols (RSNO). CysNO fate in buffer solution was evaluated by optical and electron paramagnetic resonance (EPR) spectroscopy, and the consequences on its vasorelaxant effect were studied on noradrenaline-precontracted rat aortic rings. The main results are the following: (i) copper or iron ions, especially in the presence of the reducing agent ascorbate, accelerated the decomposition of cysNO and markedly attenuated the amplitude and duration of the relaxant effect of cysNO; (ii) by contrast, the iron and copper chelators bathophenantroline disulfonic acid (BPDS) and bathocuproine disulfonic acid (BCS) exerted a stabilizing effect on cysNO, prolonged its vasorelaxant effect, and abolished the influence of ascorbate; (iii) in the presence of ascorbate, BPDS displayed a selective inhibitory effect toward the influence of iron ions (but not toward copper ions) on cysNO decomposition and vasorelaxant effect, while BCS prevented the effects of both copper and iron ions; (iv) l -cysteine enhanced stability and prolonged the relaxant effect of cysNO; (v) the process of iron-induced decomposition of cysNO was associated with the formation of EPR-detectable dinitrosyl–iron complexes (DNIC) either with non-thiol- or thiol-containing ligands (depending on the presence of l -cysteine), both of which exhibiting vasorelaxant properties. From these data, it is concluded that the amount of intrinsic copper was probably too low to produce a destabilizing effect even on the most labile RSNO, cysNO, and that only intrinsic iron, through the formation of DNIC, was responsible for the process of cysNO decomposition and thus influenced its vasorelaxant properties.

Despite short halflife in biological fluids, nitric oxide (NO) can produce remote or long lasting effect in the cardiovascular system. Long distance transport or local storage of NO might explain these effects. In blood, recent findings suggest that in addition to being a major consumption pathway, interaction of NO with hemoglobin may permit O(2)-governed transport of NO (as S-nitrosohemoglobin) to tissues in which NO may be released together with O(2), via transnitrosation of a transport protein. In blood vessels, two different putative NO stores have been characterized. The first is the photosensitive store, formed from endothelium-derived NO. The mechanism of NO release from this store in the body (in absence of light) and its physiological relevance are unknown. The second store is generated in conditions of high tissue NO levels, as a consequence of the inducible NO synthase activity or in various stress conditions. This NO store involves formation of protein-bound dinitrosyl iron complexes or S-nitrosated proteins, or both. Low molecular weight thiols can displace NO from these stores and probably transfer it to target membrane protein(s) such as K(+) channels, via transnitrosation reactions. These stores may be involved in defence mechanisms against inflammation or stress. Thus, NO transport and storage mechanisms may be implicated in a variety of NO effects. The mechanisms of their formation and of NO release and their physiologic and pathophysiologic relevance deserve further investigations.

It was demonstrated that two species of paramagnetic dinitrosyl iron complex (DNIC) with neocuproine form under the following conditions: in addition of neocuproine to a solution of DNIC with phosphate; in gaseous NO treatment of a mixture of Fe 2+ + neocuproine aqueous solutions at pH 6.5–8; and in addition of Fe 2+ − citrate complex + neocuproine to a S -nitrosocysteine (cys-NO) solution. The first form of DNIC with neocuproine is characterized by an EPR signal with g-factor values of 2.087, 2.055, and 2.025, when it is recorded at 77K. At room temperature, the complex displays a symmetric singlet at g = 2.05. The second form of DNIC with neocuproine gives an EPR signal with g -factor values of 2.042, 2.02, and 2.003, which can be recorded at a low temperature only.The revealed complexes are close to DNIC with cysteine in their stability. The ability of neocuproine to bind Fe 2+ in the presence of NO with formation of paramagnetic DNICs warrants critical reevaluation of the statement that neocuproine is only able to bind Cu + ions. It was suggested that the observed affinity of neocuproine to iron was due to transition of Fe 2+ in DNIC with neocuproine to Fe + . In experiments on cys-NO, it was shown that the stabilizing effect of neocuproine on this compound could be due to neocuproine binding to the iron catalyzing decomposition of cys-NO.

Though a large number of studies indicate that nitric oxide synthase (NOS) is responsible for NOz.rad; production in biological systems, controversy still remains concerning whether NOS directly produces NOz.rad;. Schmidt et al. (PNAS 93:144492, 1996) proposed that NOS first synthesizes nitroxyl anion (NO(-)), which is then converted to NOz.rad; by superoxide dismutase (SOD). With electron paramagnetic resonance spectroscopy using N-methyl-D-glucamine dithiocarbamate iron (Fe-MGD), we directly detected NOz.rad; from purified NOS in the absence of SOD (Xia et al., PNAS 94:12705, 1997). We also showed that the requirement for SOD in the previous NOz.rad; measurements appeared to be due to the high levels of exogenous superoxide production in their reaction system because of the presence of free FAD. However, it was recently questioned whether Fe-MGD can discriminate NOz.rad; from NO(-) (Komarov et al., FRBM 28:739-742, 2000). In this study we examined the trapping specificity of different redox forms of Fe-MGD. With Fe(2+)-MGD, NOz.rad; generated characteristic triplet NOz.rad;-Fe(2+)-MGD signals (g = 2. 04, a(N) = 12.7 G), whereas NO(-) from Angeli's salt was EPR silent. Both NOz.rad; and NO(-) gave rise to NOz.rad;-Fe(2+)-MGD signals when Fe(3+)-MGD was used. Strong NOz.rad; signals were measured from purified nNOS using the NOz.rad; selective Fe(2+)-MGD and this was not affected by SOD. Thus, spin trapping with Fe-MGD can distinguish NOz.rad; and NO(-) and this depends on the redox status of the iron. The detection of NOz.rad; from purified NOS by Fe(2+)-MGD unambiguously reconfirms our previous report that NOS directly synthesizes NOz.rad; but not NO(-).

It has been shown that a dinitrosyl dithiolato iron complex is formed under physiological conditions and that it functions as an NO transporter. In the present study, a diglutathionyl dinitrosyl iron complex [DNIC-(GS)2] was injected into mice and its abdominal distribution and behavior were examined by using electron paramagnetic resonance (EPR) spectroscopy. The X-band EPR signal intensity of the blood, liver, kidney, and spleen decreased with time but signals from the liver and kidney were readily detectable even 24h after the injection. The time courses of signal intensity were quite similar when the agent was administered via intravenous and subcutaneous injection routes, suggesting that DNIC-(GS)2 can penetrate readily and rapidly through the membranes. Real-time detection of DNIC-(GS)2 in the upper abdomen of the living mice was performed by employing an in vivo EPR spectroscopy. These results suggest that DNIC-(GS)2, an endogenous NO carrier, has an excellent membrane permeability and has a relatively high affinity for the liver and kidney.

Adaptation to hypobaric hypoxia is known to exert multiple protective effects related with nitric oxide (NO). However the effect of adaptation to hypoxia on NO metabolism has remained unclear in many respects. In the present work we studied the interrelation between NO production and storage in the process of adaptation to hypoxia. The NO production was determined by the total nitrite/nitrate concentration in rats plasma. The volume of NO store was evaluated in vitro by the magnitude of isolated aorta relaxation to diethyldithiocarbamate. It was shown that both the nitrite/nitrate level and the NO store increased as adaptation to hypoxia developed. Furthermore, the NO store volume significantly correlated with plasma nitrite/nitrate. Therefore, adaptation to hypoxia stimulates NO production and storage and these effects can potentially underlie NO-dependent beneficial effects of adaptation.

Nitric oxide (NO) content in rat cerebral cortex was measured using Electron Spin Resonance (ESR) spectroscopy. A nearly fivefold elevation in NO content was found at the peak time of pentylenetetrazole (PTZ)-induced seizures. The administration of N-nitro-l-arginine (L-NNA), a competitive inhibitor of NO-synthase, at the dose of 250 mg/kg, completely prevented the NO increase induced by PTZ, although clonic convulsions in the animals have been observed. L-NNA (10 mg/kg) was shown to delay the onset of clonic seizures as well as to shorten the latency of the first convulsive twitch. The level of lipid peroxidation secondary products measured as the content of thiobarbituric acid reactive species (TBARS) was increased in the cerebral cortex of PTZ-treated rats. L-NNA (250 mg/kg) failed to prevent the increased TBARS level produced by PTZ. The results support the notion that NO may play a trigger role in the pathophysiology of convulsive seizures.

Publisher Summary The most promising of all methods proposed so far for direct measurement of nitric oxide (NO) appears to be the use of spin traps that form stable paramagnetic adducts detectable by the electron paramagnetic resonance (EPR) method. Sodium diethyldithiocarbamate (DETC) injected into mice enters organ tissues, bound endogenous iron and forms NO traps, hydrophobic Fe 2+ -DETC that located themselves in cell membranes. When animals are injected with the compounds producing NO in the organism paramagnetic mononitrosyl iron complexes (MNIC) with DETC (MNICDETC) were formed in organs. Concentration of the complexes (and hence the amount of NO included) could be evaluated by the intensity of the characteristic EPR signal from MNIC-DETC. At present, this method of NO detection in living systems based on the use of both hydrophobic and hydrophilic iron complexes with various dithiocarbamate derivatives is of great utility in investigations on NO biology. This chapter discusses the physicochemical properties of mononitrosyl iron complexes (MNIC) - sodium diethyldithiocarbamate (DETC). Some peculiarities of MNIC-DETC formation, and methodological aspects and results of using the Fe 2+ DETC complexes for detection, and quantitative determination of nitric oxide (NO) formed in animal tissues and cell cultures from endogenous sources. The MNIC-DETC solved in organic polar solvents can be reversibly oxidized by halogens and nitrogen dioxide (NO 2 ) and transformed into the diamagnetic state with the iron electronic configuration. The X-ray analysis of a reaction product of MNIC-DETC and NO 2 taken as an example showed that oxidizing agents incorporate into this complex at the cis -position to NO.

The parameters of EPR signal from dinitrosyl-iron complexes (DNIC) with bovine serum albumin (BSA), horse hemoglobin (Hb), and apometallothionein (apo-Mt) of horse kidney incorporating one (BSA, Hb) or two thiol-containing ligands (apo-Mt) were compared. The EPR signal from DNIC-BSA was characterized by the rhombic symmetry of g tensor at room temperature of signal recording (ambient temperature) or at 77K in the solution frozen in the presence of glycerol. In freezing of the solution in the absence of glycerin, under the exposure of DNIC-BSA to negatively charged sodium dodecyl sulfate (SDS) ions, or in the incorporation of DNIC-BSA into the reversed micelles formed by negatively charged ions of surfactant aerosol OT, the symmetry of the g tensor of DNIC-BSA EPR signal increased to axial. A similarly high symmetry of g tensor was observed for the DNIC-Hb EPR signal in the absence of any influence on this protein complex. The shape of EPR signals from these preparations recorded at 77K was identical to that of EPR signal from DNIC with cysteine in frozen solution. In this connection it was concluded that the EPR signal from this low-molecular DNIC with the (RS-)2Fe+(NO+)2 structure cannot be considered as a peculiar "fingerprint" of DNIC with the same structure in biosystems. In such systems the same signal can originate from protein DNIC incorporating only one thiol-containing ligand along with a nonthiol ligand. The EPR signal displayed by DNIC with apo-Mt with a high content of cysteine residues at room temperature of registration was identical to the EPR signal from frozen solution of DNIC with cysteine. This protein DNIC is apparently characterized by the same structure as DNIC with cysteine.

The NO + Fe2+ + thiols system in an aqueous solution has been found earlier to exhibit temporal oscillatory changes in the concentration of paramagnetic dinitrosyl iron complexes with thiol-containing ligands and S-nitrosothiols, as well as in the concentration of free iron (not included in the complexes). It is proposed that autowaves can appear in this system characterized by periodic changes in the concentrations of its components in time and space. Such changes may form a basis for the control of the physiological effects of nitric oxide, dinitrosyl iron complexes, and S-nitrosothiols as agents affecting various cellular and tissue targets.

Formation of S-nitrosothiols was demonstrated in 1-50 mM aqueous solutions of cysteine or glutathione (cys-NO or GS-NO, respectively) upon contact of thiols with gaseous nitric oxide under a pressure of 50-600 mm Hg and anaerobic conditions. The yield of S-nitrosothiols was increased by mixing with NO plus air at a molar ratio [NO]/[O2 from air] of no less than 40. In this instance, the S-nitrosothiol formation was optimum at a NO pressure of 100-150 mm Hg. The addition of 0.25 mM o-phenanthroline, a selective Fe2+ chelator, to thiol solutions prior to the treatment with NO or NO + air completely blocked the formation of S-nitrosothiols. On the other hand, this process was potentiated by the addition of Fe2+ but not Cu2+ ions. These data indicated a crucial influence of Fe2+ on the process. The contact of o-phenanthroline with S-nitrosothiols synthesized by a routine method (treatment of thiol solutions with the NO + NO2 mixture at pH

A variety of magnetic adsorbents for different biomedical applications have been developed. The adsorbents are fine disperse particles, consisting of a ferromagnetic core made of iron or its oxides, and attached to a carbon structure or coated with carbon. The size of the particles is 0.01 to 1 µm. The carbon phase is porous, has a considerable specific surface and is a good adsorbent. It can be manufactured with selective sorption properties. The ferromagnetic core allows the use of a magnetic field to guide and move the particles and regulate their agglomeration behavior. The particles were produced by three different methods, and their sorption properties, shape, size distribution, magnetic and biological properties were studied. The particles were successfully used in several biomedical applications, including magnetic localization of anti-cancer drugs in the tumor zone. Magnetic targeting allows to create a hundred times higher concentrations of anti-cancer antibiotics in the tumor compared with other organs, which is maintained for 8 to 10 days after a single injection. Animal and pre-clinical trials have been completed and permission for clinical trials on cancer patients with high grade disease was obtained. Since 1990, more than a hundred patients with different types of tumors have been treated in several Russian clinics. Many patients had either full recovery or their condition was considerably improved.

Nitric oxide (NO), a highly reactive, short-lived radical, is believed to be a trans- neuronal messenger in the central nervoussystem. NO is generated by the enzyme NO- synthase, which was shown to occur widely in the central nervous system. NO has been implicated in several brain disorders such as Alzheimer’s and Parkinson’s deseases, stroke, trauma, epilepsy, etc. Under these pathological conditions excessive release of excitatory amino acid glutamate results in increase of NO content in the brain to a toxic level. Epilepsy is one of the diseases in which excitatory amino acids are implicated and NO could be an important pathogenetic factor in the mechanisms that underlie seizure induction, propagation and progression. However, acute effects of neuronal NO overflow on brain functions are still a matter of controversy. NO-synthase, the enzyme responsible for NO synthesis in mammalian brain from L-arginine, can be blocked by arginine analogues such as L-NNA. These compounds can be used as a tool to investigate the role of nitric oxide in various cellular mechanisms. Recent reports claim NO to be either pro- or anticonvulsant (1,2). The aim of the present study was to prove whether model convulsive states of different origin in rats may involve any alteration in NO content and in content of secondary products of lipid peroxidation in the brain cortex.

ESR-spectrometry was used to investigate radiation-induced paramagnetic centers in enamel of mammals: carnivores (polar bear and fox), ungulates (reindeer, European bison, moose), and man. Values at half the microwave power saturation of the radiation signal, P1/2, evaluated at room temperature, was found to range from 16 to 26 mW for animals and man. A new approach to discrimination of the radiation induced signal from the total ESR spectrum of reindeer enamel is proposed. 'Dose-response' dependencies of enamel of different species mammals were measured within the dose range from 0.48 up to 10.08 Gy. Estimations of 'radiosensitivity' enamel of carnivores and ungulates showed good agreement with radiosensitivity enamel of man by ESR method.

The objective of this study was to identify a potential mechanism for S-nitrosation of proteins. Therefore, we assessed S-nitrosation of bovine serum albumin by dinitrosyl-iron-di-L-cysteine complex [(NO)2Fe(L-cysteine)2], a compound similar to naturally occurring iron-nitrosyls. Within 5-10 min, (NO)2Fe(L-cysteine)2 generated paramagnetic albumin-bound dinitrosyl-iron complex and S-nitrosoalbumin in a ratio of 4:1. Although S-nitroso-L-cysteine was concomitantly formed in low amounts, its concentration was not sufficient to account for formation of S-nitrosoalbumin via a trans-S-nitrosation reaction. Low oxygen tension did not affect S-nitrosation by the dinitrosyl-iron complex thus excluding the involvement of oxygenated NOx-species in the nitrosation reaction. Blockade of albumin histidine residues by pyrocarbonate, which prevented formation of dinitrosyl-iron-albumin complex, did not inhibit S-nitrosation of albumin. Thus, S-nitrosation of albumin by (NO)2Fe(L-cysteine)2 can proceed by direct attack of a nitrosyl moiety on the protein thiolate, without previous binding of the iron. We conclude that protein-bound dinitrosyl-iron complexes detected in high concentrations in certain tissues provide a reservoir of S-nitrosating species, e.g. low molecular dinitrosyl iron complexes.

When injected into mice prior to the NO generation increase induced with lipopolysaccharide (LPS) from Escherichia coli, exogenous antioxidants diethyldithiocarbamate (DETC) or phenazan (sodium 3.5-di- tert -butyl-4-oxiphenylpropionate) as well as the inhibitor of protein biosynthesis, cycloheximide (CHI) attenuated the NO production in mouse liver in vivo. These data demonstrated the key role of free radicals, which were likely, active oxygen species, in the synthesis of inducible NO-synthase (iNOS) responsible for the NO production in this organ. Similar effects of phenazan and CHI were observed in livers of mice treated with γ-irradiation or LPS + Fe 2+ -citrate, which suggested that these treatments also induced 1NOS synthesis through initiating the action of active oxygen species. The rate of NO synthesis was estimated by accumulation of paramagnetic mononitrosyl iron complexes with DETC (MNIC-DETC) detected using the EPR method. The formation of MNIC-DETC complexes was found in the brain of mice pre-treated with LPS + Fe 2+ -citrate which seemed to be due to iNOS synthesis stimulated by this treatment.

We identified the source of the nitrogen included into nitric oxide (NO) and studied the relationship between formation of NO, intracellular dinitrosyl ferrous iron complex (DNIC) and release of nitrite by murine bone-marrow-derived macrophages stimulated with E. coli lipopolysaccharide (LPS). NO was trapped in the cell membrane by iron-diethyldithiocarbamate complex (FeDETC) and was detected as a paramagnetic NOFe(DETC)2 complex by electron paramagnetic resonance (EPR) spectroscopy. Macrophages stimulated for 7 h up to 48 h with LPS and then incubated for 2 h with DETC exhibited an anisotropic EPR signal of axial symmetry with g- factor values g ⊥ = 2.035 , g ∥ = 2.02 and a triplet hyperfine structure (hfs) at g ⊥ characteristic of NOFe(DETC)2. In cells incubated with [15NG] l -arginine instead of [14NG] l -arginine the EPR signal of [15N]OFe(DETC)2 was detected with a doublet hfs at g ⊥ , indicating that NO was generated exclusively from the terminal guanidino-nitrogen of extracellular l -arginine. The ratio of NO formation and of nitrite release changed with time of exposure to LPS, nitrite exceeding NO at early stages of macrophage activation, and NO exceeding nitrite at later stages. DNIC with thiolate ligands (0.5 nmol/107 cells) was observed in stimulated macrophages not loaded with DETC. Furthermore, DNIC released from macrophages was trapped in the extracellular medium by bovine serum albumin (BSA) (1 nmol/107 cells per 2 h) by formation of a paramagnetic DNIC with BSA. DNIC release not only provides a route for iron loss from activated macrophages, but may also play a role in the cytotoxic and microbiostatic activity of macrophages.

Administration of an Fe 2+ -citrate complex to mongrel mice pretreated with lipopolysaccharide (LPS) from Salmonella typhosa increased LPS-induced NO formation in vivo in the liver, intestine, lung, heart, kidney and spleen by 10–20-fold. This process was monitored by the intensity of the EPR signal due to mononitrosyl iron complex (MNIC) formation with exogenous diethyldithiocarbamate (DETC) recorded in the tissues. The NO synthase inhibitor, N G - nitro- l -arginine , prevented this complex formation in the liver of mice treated with both LPS and Fe 2+ -citrate complex. Thus, administration of LPS and Fe 2+ -citrate complex to mice induced NO biosynthesis in this tissue via an l -arginine-dependent pathway, presumably by facilitating the entry of Ca 2+ ions into NO-producing cells through Fe 2+ -induced cell membrane lesions.

In cultured macrophages (J 774 line) a decrease in iron-sulfur centers (ISC) was not observed after 5 min treatment with nitric oxide (NO) (10(-7) M NO/10(7) cells). The content of these centers was measured by electron spin resonance (ESR) spectroscopy at 16-60 K. However, the appearance of a characteristic ESR signal at g(av) = 2.03 indicated the formation of dinitrosyl iron complex (DNIC) in these cells. These findings suggest that loosely bound non-heme iron (free iron) but not iron from ISC is mainly involved in DNIC formation. ISC might release iron for DNIC formation after their destruction induced by the products of NO oxidation (NO2, N2O3, etc).

The effect of diethyldithiocarbamate (DETC, 10−3 M) on the vasorelaxant activity of acetylcholine, nitric oxide (NO), nitrite, glyceryl trinitrate or dinitrosyl iron cysteine complexes was studied in isolated rat aortic rings contracted with norepinephrine. Pretreatment of these segments with DETC attenuated the vasorelaxation induced by vasodilators and prevented the subsequent restoration of vessel tone, whereas DETC added after the vasodilators induced a rapid restoration of tone. The inhibitory effect of DETC was due to the trapping of NO by a complex of DETC with Fe2+ formed in the tissue.

The most recent data on mechanisms of spin trapping of nitric oxide (NO) by iron dithiocarbamate complexes in animal and plant cells and tissues are considered. The rationale is as follows: 1 In the absence of NO in cells and tissues, iron binds primarily to compounds others than dithiocarbamate ligands, e.g., tricarbonic acids. 2. Predominant binding of iron to dithiocarbamate ligands takes place only after its binding to NO, since nitrosylated iron manifests much higher affinity for these ligands that for any non-thiol compounds. 3.Within the composition of mononitrosyl dithiocarbamate complexes, iron exists predominantly in the oxidized (Fe3+) form, i.e., these complexes are originally diamagnetic. Their subsequent single-electron reduction to the paramagnetic, EPR -detectable form is mediated by endogenous or exogenous (e.g., dithionite) reducing agents. 4.Superoxide-mediated transition of paramagnetic mononitrosyl dithiocarbamate iron complexes into EPR-silent state can be accompanied by significant reduction of EPR-detectable complexes. This defect can be overcome through the use of the so-called ABC method. 5. In contrast to hydrophobic complexes fast decomposition of water-soluble mononitrosyl iron complexes in animal organisms testifies to low efficiency of these complexes in determination of NO content in animal cells and tissues.

We studied the biological activity, stability and interaction of dinitrosyl-iron(II)-L-cysteine with vascular tissue. Dinitrosyl-iron((II)-L-cysteine was a potent activator of purified soluble guanylyl cyclase (EC50 (nM with and 100 nM without superoxide dismutase) and relaxed noradrenaline-precontracted segments of endothelium-denuded rabbit femoral artery (EC50 10 nM superoxide dismutase). Pre-incubation (5 min; 310 K) of endothelium-denuded rabbit aortic segments with dinitrosyl-iron(II)-L-cysteine (0.036–3.6 mM) resulted in a concentration-dependent formation of a dinitrosyl-iron(II complex with protein thiol groups, as detected by ESR spectroscopy. While the complex with proteins was stable for 2 h at 310 K, dinitrosyl-iron(II)-L-cysteine in aqueous solution (30–360 μM) decomposed completely within 15 min, as indicated by disappearance of its isotropic ESR signal at gav = 2.03 (293 K). Aortic segments pre-incubated with dinitrosyl-iron(II)-L-cysteine released a labile vasodilating and guanylyl cyclase activating factor. Perfusion of these segments with N-acetyl-L-cysteine resulted in the generation of a low molecular weight dinitrosyl-iron(II)-dithiolate from the dinitrosyl-iron(II) complex with proteins, as revealed by the shape change of the ESR signal at 293 K. The low molecular weight dinitrosyl-iron(II)-dithiolate accounted to an enhanced guanylyl cyclase activation and vasodilation induced by the aortic effluent. We conclude that nitric oxide (NO) produced by, or acting on vascular cells can be stabilized and stored as a dinitrosyl-iron(II) complex with protein thiols, and can be released from cells in the form of a low molecular weight dinitrosyl-iron(II)-dithiolate by intra- and extracellular thiols.

The reduction of adrenal ferredoxin (adrenodoxin) at low temperatures was investigated in order to separate local modifications of the active centre of the protein on its reduction, from the conformational transition which seems to accompany the change of the redox state of the irons. The ESR spectra of the states of the protein, where the reduced active centre is to be found by the “oxidized” conformation of the apoprotein, were obtained. The transition from the states of the protein to the state which occurs on its chemical reduction at room temperature was also investigated. The results of the work support the view that conformational changes in proteins (enzymes) which take place while they are functioning proceed after modifications of the active centres (change of the redox state, adsorption of a substrate, etc.), and are essentially caused by them. Adrenal ferredoxin was the third subject in our studies of the intermediate states of proteins which appear after reduction of their active centres by means of electrons trapped in water-ethylene glycol mixtures at the temperature of liquid nitrogen [1, 2]. In the reduced state, the active centre of the protein has an ESR signal with a g-factor of 1.94 [3, 4] which is convenient for our purposes.

It has thus been shown that the reduction of heme-containing proteins does in fact pass through two main stages, the first of which does not require significant conformation rearrangement. This means that the rearrangement of the environment may take place after the electron reaches the appropriate orbital of the active center. The kinetic stabilization method has made it possible to detect the thermodynamically nonequilibrium intermediate states and in one case (adrenodoxin) to follow their subsequent relaxation in detail. In the case of hemoglobin it is possible to detect the low-spin reduced state, which has not been observed before. It may be assumed that the nonequilibrium states and the structural relaxation play an important role not only in proteins, but also in other reactions in the condensed phase. There are grounds for assuming that the proposed method of recording intermediate states will prove useful for the investigation of the mechanism of these processes. It should also be noted that by converting the active center from the oxidized to the reduced form without change in the structure of the environment it has been possible to reach a number of new conclusions regarding the structure of the equilibrium oxidized states. This technique may be of some methodological importance.

The ability of nitric oxide (NO) donor compounds to induce the SOS DNA repair response in Escherichia coli is reported. Dinitrosyl iron complexes with glutathione and cysteine (DNIC) are the most potent SOS-inducers. S -Nitrosothiols (RSNO) mediate a similar response at 10–100 μM, but the response decreases sharply at concentrations above 0.5 mM. Pretreatment of the cells with the chelating agent o -phenanthroline (OP) prevents induction of the SOS response by all agents used. On the other hand, the toxicity of S -nitrosothiols is higher than that of DNIC. The EPR study showsthe appearance of an EPR DNIC-type signal after incubation of the cells with S -nitrosoglutathione because of mutual transformation between RSNO and DNIC in the presence of accessible iron inside the cells. Pretreatment of the cells with OP leads to a decrease in this signal. Analysis of NO donor effects reveals a dual role of the iron ions in reactivity and toxicity of the compounds studied, i.e. (i) stabilization of the cytotoxic RSNO and (ii) generation of the SOS signal.

The concentration-dependent effect of various nitric oxide donors on synthesis of different heat shock proteins was evaluated in Reuber H35 hepatoma cells and their heat shock protein-inducing ability was compared with the effect of a heat shock. A 6 h incubation of H35 cells with the dimeric (diamagnetic) form of dinitrosyl iron complex with glutathione or N-acetyl-L-cysteine activated synthesis of various heat shock proteins, heat shock protein 28, 32, 60, 70, 90 and 100. Synthesis of these proteins was evaluated by [35S]methionine and [35S]cysteine labelling with subsequent separation of proteins by polyacrylamide gel electrophoresis. The dinitrosyl iron complex with glutathione appeared to be the most efficient inductor of heat shock protein synthesis and initiated the synthesis of heat shock protein 28 even more efficiently than a 30 min heating of cells. In the same experiments, S-nitroso-D,L-penicillamine exerted a considerably lesser effect on the synthesis of heat shock proteins. It was suggested that the active moiety of dinitrosyl iron complexes as inductors of heat shock protein synthesis is represented by their Fe+(NO+)2 groups which move to thiol groups of the proteins participating in the initiation of heat shock protein synthesis.