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This study aimed to examine whether inducible nitric oxide synthase (iNOS) plays a role in the delayed antiarrhythmic effect of sodium nitrite. Twenty-one dogs were infused intravenously with sodium nitrite (0.2 μmol·kg -1 ·min -1 ) for 20 min, either in the absence (n = 12) or in the presence of the iNOS inhibitor S-(2-aminoethyl)-isothiourea (AEST) (total dose 2.0 mg·kg -1 i.v., n = 9). Control dogs (n = 12) were given saline. Twenty-four hours later, all of the dogs were subjected to a 25 min period occlusion of the left anterior descending coronary artery followed by rapid reperfusion. Dogs treated with AEST and nitrite received again AEST prior to the occlusion. Compared with the controls, sodium nitrite markedly reduced the number of ectopic beats, the number and incidence of ventricular tachycardia, and the incidence of ventricular fibrillation during occlusion and increased survival (0% versus 50%) from the combined ischaemia and reperfusion insult. Although AEST completely inhibited iNOS activity, the nitrite-induced increase in NO bioavailability during occlusion was not substantially modified. Furthermore, AEST attenuated but did not completely abolish the antiarrhythmic effect of nitrite. The marked delayed antiarrhythmic effect of sodium nitrite is not entirely due to the activation of iNOS; other mechanisms may certainly play a role.

This unit describes a procedure for separating T cells from other mononuclear cells by exploiting the unique ability of cells to bind to and form rosettes with sheep red blood cells (SRBC). This isolation method also allows recovery of the nonrosetting cell population (B lymphocytes, monocytes, and macrophages). Neuraminidase- and 2-aminoethylisothiouronium bromide (AET)-treated SRBC are used for rosetting because of enhanced binding to T cells. It should be noted that use of the rosetting technique to obtain purified T cells or purified non-T cells by negative selection has largely been superceded by other techniques such as panning and immunomagnetic beads., (Copyright © 2016 John Wiley & Sons, Inc.)

Background and Objectives: Intravascular haemolytic reactions are reported in red-cell T-activated patients after blood transfusion. The relationship between T antigen antibodies present in normal plasma and these reactions remains unclear. In this study, we assessed the haemolytic activity of T antibodies in vitro in comparison with anti-A/B antibodies., Materials and Methods: We established a haemolysis assay based on treating target red-blood-cells (RBCs) with 2-aminoethylisothiouronium bromide (AET). Two hundred and seven blood donor sera were analysed for anti-T, anti-A/B haemolysins and anti-T agglutinins., Results: Anti-T haemolysins were found in 4 (1·9%) blood donor sera using a standard haemolysis method and in 174 (84%) samples using AET-treated RBCs. Haemolysis correlated with agglutination titres (P<10(-7) ). With both methods, anti-T haemolysins were much weaker than anti-A and anti-B haemolysins. Gradual desialylation of RBCs showed a correlation between sialic acid level as indicated by agglutination with Sambucus nigra lectin and anti-T mediated haemolysis that was significantly increased (fold 2·4) independently of T antigen expression., Conclusion: These data indicate that, in vitro, anti-T-mediated haemolysis depends primarily on the degree of desialylation of target RBCs. They suggest that the haemolytic activity of T antibodies-containing human sera is usually weak and may only become significant in the very rare setting of a profound desialylation of RBCs., (© 2010 The Author(s). Vox Sanguinis © 2010 International Society of Blood Transfusion.)

This study demonstrates that aminoethylisothiourea (AET), a potent inhibitor of inducible nitric oxide synthase, is an irreversible competitive inhibitor of mushroom tyrosinase by chelation to the active site of tyrosinase when L-3,4-dihydroxyphenylalanine was assayed spectrophotometrically. The spectrophotometric recordings of the inhibition of tyrosinase by AET were characterized by the presence of a lag period prior to the attainment of an inhibited steady-state rate. The lag period corresponded to the time in which AET was reacting with the enzymatically generated o-quinone. Increasing AET concentrations provoked longer lag periods as well as a concomitant decrease in the tyrosinase activity. Both lag period and steady-state rate were dependent on AET, substrate, and tyrosinase concentrations. The inhibition of diphenolase activity of tyrosinase by AET showed positive kinetic cooperativity which arose from the protection of both substrate and o-quinone against inhibition by AET. The UV-visible spectrum of a mixture of tyrosinase and AET exhibited a characteristic shoulder peak ascribed to the chelation of AET to the active site of tyrosinase. Moreover, the presence of copper ions only partially prevented but not reverted mushroom tyrosinase inhibition when CuSO(4) was added to the assay medium on tyrosinase activity.

The aim of this study was to assess radioprotective effects of quercetin and the ethanolic extract of propolis (EEP) in CBA mice exposed to a single radiation dose 4 Gy (60Co). The mice were treated with 100 mg kg(-1) quercetin or EEP a day for three consecutive days either before (pre-treatment) or after gamma-irradiation (therapy). Leukocyte count was determined in blood drawn from the tail vein, and DNA damage in leukocytes was assessed using the alkaline comet assay. Genotoxic effects of the test compounds were also evaluated in non-irradiated mice. The levels of radioprotection provided by both test compounds were compared with those established in mice that were given chemical radioprotector S-(2-aminoethy1)isothiouronium bromide hydrobromide (AET). Mice that received pre-treatment were less sensitive to irradiation. Mice given the post-irradiation therapy showed a slight but not significant increase in total leukocyte count over irradiated negative control. Quercetin showed better protective properties than EEP in both pre-treatment and therapy, and activated a higher number of leukocytes in non-irradiated mice. The alkaline comet assay suggests that both natural compounds, especially when given as pre-treatment, protect against primary leukocyte DNA damage in mice. At tested concentrations, EEP and quercetin were not genotoxic to non-irradiated mice. AET, however, caused a slight but not significant increase in DNA damage. Although the results of this study show the radioprotective potential of the test compounds, further investigation is needed to clarify the underlying protection mechanisms.

This study investigated the role of nitric oxide in radiation-induced damage by examining changes in mouse serum nitrate concentrations after irradiation. In addition, the contribution of S-2-aminoethylisothiourea 2HBr (AET) to the mechanisms of radiation damage protection was also clarified. The serum nitrate concentration increased as soon as 1.5 h after irradiation, and after 2.5 to 3.0 h the concentrations were significantly higher compared with normal levels. Normal levels were re-established after 12 h. Post-irradiation serum nitrate concentrations increased dose-dependently with irradiation dose (19.6-31.5 Gy). AET suppressed increases in the serum nitrate concentration following irradiation while 2-mercaptoethylamine HCl (MEA) did not. AET has an inhibitory effect on inducible nitric oxide synthase (iNOS); therefore, the increase in nitric oxide after irradiation may be produced by iNOS. Combined administration of irradiation and lipopolysaccharide (LPS) induced a significant increase in serum nitrate concentration, and a significant decrease in survival rate, compared with irradiation alone. The administration of AET or aminoguanidine increased survival rate following irradiation. In contrast to findings after LPS administration, IL-1beta and IFN-gamma were not determined in serum following irradiation. Existing iNOS is activated by irradiation, and nitric oxide production appears to increase without iNOS induction. Thus, the irradiation-induced increase in nitric oxide may be related to lethal injury.

We studied the role of extracellular and intracellular NO in the regulation of the stress response and apoptosis in macrophages of proinflammatory and antiinflammatory phenotypes under the influence of S. aureus and heat shock. Blockade of extracellular nitric oxide synthesis in cells with antiinflammatory phenotype inhibited the stress response induced by S. aureus and heat shock. The decrease in extracellular nitric oxide concentration around antiinflammatory macrophages potentiated the stress response induced by S. aureus, but had no effect on the stress response induced by heat shock. Hence, intracellular NO mediates the stress response induced by S. aureus and heat shock, while extracellular NO inhibits the stress response induced by S. aureus, but has no effect on the stress response induced by heat shock. In cells with antiinflammatory phenotype, intracellular NO plays an antiapoptotic role. S. aureus and heat shock did not cause apoptosis in macrophages with proinflammatory phenotype, while intracellular NO did not play a role in antiapoptotic activity of the proinflammatory phenotype. Extracellular NO synthesized by macrophages protects these cells from apoptosis induced by S. aureus and heat shock.

In this study, we examined the regulation by putrescine, spermidine and spermine of nitric oxide (NO) biosynthesis in Arabidopsis thaliana seedlings. Using a fluorimetric method employing the cell-impermeable NO-binding dye diaminorhodamine-4M (DAR-4M), we observed that the polyamines (PAs) spermidine and spermine greatly increased NO release in the seedlings, whereas arginine and putrescine had little or no effect. Spermine, the most active PA, stimulated NO release with no apparent lag phase. The response was quenched by addition of 2-aminoethyl-2-thiopseudourea (AET), an inhibitor of the animal nitric oxide synthase (NOS) and plant NO biosynthesis, and by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-1-oxy-3-oxide (PTIO), an NO scavenger. By fluorescence microscopy, using the cell-permeable NO-binding dye diaminorhodamine-4M acetoxymethyl ester (DAR-4M AM), we observed that PAs induced NO biosynthesis in specific tissues in Arabidopsis seedlings. Spermine and spermidine increased NO biosynthesis in the elongation zone of the Arabidopsis root tip and in primary leaves, especially in the veins and trichomes, while in cotyledons little or no effect of PAs beyond the endogenous levels of NO-induced fluorescence was observed. We conclude that PAs induce NO biosynthesis in plants.

We have shown previously that a single period of treadmill exercise in dogs protects the heart against the severe ventricular arrhythmias that arise when a major (anterior descending) branch of the left coronary artery is occluded following anaesthesia 24 h later. This protection is aminoguanidine sensitive, suggesting a role for nitric oxide (NO) in this exercise-induced delayed antiarrhythmic effect. The present study has further examined the possible role of NO as a mediator and/or as a trigger using the selective induced (iNOS) inhibitor S-(2-aminoethyl)-methyl-isothiourea (AEST) and the specific but not selective nitric oxide synthase inhibitor N(omega)-nitro-L-arginine-methyl-ester (L-NAME). Exercise markedly reduced the severity of ischaemia and reperfusion-induced ventricular arrhythmias 24 h later. Thus, only one of the dogs (8%) so exercised fibrillated on occlusion (contrast 46% in the control, non-exercised dogs; P<0.05) and the marked changes in the inhomogeneity of electrical activation that occur in the ischaemic region following occlusion were much reduced (P<0.05 compared to controls). This delayed exercise-induced cardioprotection was significantly attenuated by the nitric oxide synthase (NOS) inhibitors L-NAME, given prior to the exercise protocol and by AEST given prior to the coronary artery occlusion. For example, survival from the ischaemia-reperfusion insult was 54% in the exercise dogs, 0% in the controls and 14% in those dogs given a NOS inhibitor. We conclude that nitric oxide (NO) is both the trigger and the mediator of this delayed protection against ischaemia and reperfusion-induced arrhythmias.

Inducible nitric oxide synthase (iNOS) is one of the clinical targets in rheumatoid arthritis. Synoviocytes, macrophages, and chondrocytes in the joints of patients with rheumatoid arthritis appear to express iNOS, but the contribution of iNOS molecules to rheumatoid arthritis is not yet clear. This study used adjuvant-induced arthritis in rats as a model to examine the association between the iNOS expression and its activity in rheumatoid arthritis. In adjuvant-injected rats, arthritic changes in the paw were first observed between days 10 and 12. NO-generation activity was precisely determined by combining an electron spin resonance/nitric oxide (NO)-trapping method with the method of standard addition using an NO generator, and we found that the activity in the joint samples was extremely high on day 10. The administration of S-(2-aminoethyl)isothiourea, a selective iNOS inhibitor, from day 0 to day 10, effectively reduced the paw swelling. Immunohistological studies showed that chondrocytes expressed iNOS on days 7-14 and that nitrotyrosine residues, a footprint of NO generation, were produced on day 10. This indicates that NO generation by iNOS induced in chondrocytes is a key event in the induction of adjuvant arthritis.

Capability of S-radioprotectors (AET, 2-AT, 2-ADT) to react with OH-radicals and to protect various molecular biotest systems against radiation damage was compared with that of 5-methoxytryptamine, some amino acids and t-butanol. A method of competing acceptors was used to determine rate constants in reactions of the radioprotectors with OH-radicals. A complex of biotest systems (protein, DNA, protein-lipid complex) was applied to estimate the radioprotective activity in vitro. It was found that the studied S-compounds are capable of modifying the protective effect as compared to the expectation from the competitive kinetics approach. Both enhancing and lessening of the effect was observed depending on the test system used. The obtained results can be explained by the impact of secondary radicals on the bio-target and/or by the interaction of the S-compounds with the bio-target that altered its radiosensitivity.

A model to study the effectiveness of potential chemopreventive agents that inhibit neoplastic process by different mechanisms has been used to test the efficacy of seven nitric oxide synthase (NOS) inhibitors. Five selective inducible NOS (iNOS) inhibitors: S-methyl isothiourea (S-MITU), S-2-aminoethyl isothiourea (S-2-AEITU), S-ethyl isothiourea (S-EITU), aminoguanidine (AG), 2-amino-4-methyl pyridine (2-AMP), and two non selective general NOS inhibitors: l-N(6)-(1-iminoethyl) lysine (IEL) and N(omega)-nitro-l-arginine (NNLA), were tested for efficacy against a carcinogen, benzo[a]pyrene (B[a]P)-induced primary rat tracheal epithelial (RTE) cell transformation assay. RTE cells were treated with B[a]P alone or with five nontoxic concentrations of an NOS inhibitor and the resulting foci at the end of 30 days were scored for inhibition of transformation. The results indicate that all three isothiourea compounds inhibited B[a]P-induced RTE foci in a dose-dependent manner. S-AEITU was the most effective inhibitor with an IC(50) (the molar concentration that inhibits transformation by 50%) of 9.1 microM and 100% inhibition at the highest dose tested (30 microM). However, both S-EITU and S-MITU showed a maximum percent inhibition of 81% and 100% at 1 mM with an IC(50) of 84 and 110 microM, respectively. 2-AMP did not show any dose-dependent response, but was highly effective (57% inhibition) at an intermediate dose of 30 microM and an IC(50) of 25 microM. Similar to thiourea compounds, AG exhibited good dose-dependent inhibition with a maximum inhibition of 86% at 1 mM. NNLA and IEL were negative in this assay. Based on the IC(50) values, NOS inhibitors were rated for efficacy from high to low as follows: S-2-AEITU<2-AMP

Nitric oxide (NO) produced by macrophages is thought to contribute to various pathological conditions. Osteopontin (OPN) is a phosphorylated glycoprotein produced principally by macrophages. OPN inhibits inducible nitric oxide synthase (iNOS), which generates large amounts of NO production. However, the relationship between NO and endogenous OPN in activated macrophages has not yet been elucidated. We therefore examined expression of endogenous iNOS and OPN in a murine macrophage cell line, RAW 264.7 cells, by treating the cells with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Treatment of cells with LPS and IFN-gamma resulted in an increase of iNOS mRNA to maximum at 12 h after stimulation. In contrast, OPN mRNA was induced more slowly than iNOS mRNA. Induction of both iNOS and OPN mRNA in RAW 264.7 cells was markedly suppressed by addition of the specific iNOS inhibitor S-2-aminoethyl isothiourea dihydrobromide. The NOS inhibitor NG-methyl-L-arginine also suppressed induction of OPN mRNA but hardly affected iNOS mRNA expression. The NO-releasing agent spermine-NONOate but not peroxynitrite enhanced induction of OPN mRNA. These results suggest that NO directly up-regulates the endogenous OPN in macrophages stimulated with LPS and IFN-gamma. This up-regulation of endogenous OPN may represent a negative feedback system acting to reduce iNOS expression.

Objective: To examine the effects of the inducible nitric oxide synthase inhibitor aminoethyl-isothiourea (AE-ITU) on haemodynamic measurements, and correlate these with hepatic morphology and function in a porcine model of endotoxaemia., Design: Experimental study., Animals: 15 juvenile pigs., Interventions: Flow probes were placed around the hepatic artery and portal vein. Catheters were introduced into the portal and hepatic veins, pulmonary artery, and aorta. Infusion of AE-ITU was started one hour before that of endotoxin (study group n = 6); thereafter both substances were infused simultaneously until the end of the study (6 hours). The controls (n = 9) had endotoxin alone., Main Outcome Measures: Hepatic morphology assessed by light and electron microscopy; and hepatic integrity and function by transaminase activities and oxygen consumption. Systemic, pulmonary, and hepatic blood flow and pressure., Results: AE-ITU maintained systemic blood pressure (p < 0.05 compared with controls) without causing pulmonary hypertension. Neither hepatic morphology nor function were adversely influenced., Conclusion: In endotoxaemia AE-ITU has a favourable haemodynamic profile which is achieved without impairment of hepatic function or morphology.

Right ventricular pacing in lightly anaesthetized dogs (4x5 min periods at a pacing rate of 220 beats/min) protects against the consequences of coronary artery occlusion when this is initiated 24 h after the pacing stimulus. The main purpose of the present experiments was to determine whether repeating the pacing stimulus, at a time when protection from the initial stimulus had faded (48 h), prolonged the protection afforded against ischaemia-induced ventricular arrhythmias and other ischaemic changes (epicardial ST-segment mapping; changes in the degree of electrical inhomogeneity in the ischaemic region). Dogs were paced on two occasions, with a 48 h period between and, at different times (48, 72 and 96 h) after the second pacing stimulus, were re-anaesthetized and subjected to occlusion of the left anterior descending coronary artery. There was a marked reduction in the severity of ischaemia-induced arrhythmias 48 and 72 h after the second pacing stimulus (reduction in occlusion-induced and reperfusion-induced ventricular fibrillation, e.g. at 72 h 0/11 during occlusion and only 3/11 following reperfusion, compared to 7/21 and 10/21 respectively in the controls P<0.05). The protection had disappeared 96 h following the second pacing stimulus. Changes in ST-segment elevation and in the degree of inhomogeneity largely followed these changes in the severity of ventricular arrhythmias. The results suggest the possibility of maintaining protection against life-threatening arrhythmias following coronary occlusion by repeating a preconditioning pacing stimulus. We also demonstrate that this prolonged protection afforded by repeated cardiac pacing is mediated by nitric oxide, since the marked antiarrhythmic effect observed, e.g. 72 h after the second pacing stimulus, was abolished when S-(2-aminoethyl)-isothiourea (AEST), a particularly selective inhibitor of iNOS, had been administered before coronary artery occlusion., (Copyright 1999 Academic Press.)

The effect of sodium diethyldithiocarbamate (DDC) and S-2-aminoethyl-isothiouronicadenosin-5-triphosphate (adeturon) in the induction of Escherichia coli SOS response promoted by gamma-irradiation was studied by measuring the induction of sulA gene and the induction of lambda prophage. Furthermore, as a way of measure the exonuclease activity in gamma-irradiated cells in the presence or absence of both compounds, the DNA degradation was determined. Adeturon did not affected DNA degradation, but inhibited the induction of the SOS functions studied. On the contrary, DDC inhibited DNA degradation as well as the induction of the sulA gene, but enhanced lambda induction in E. coli lysogenic strains. These results indicate that both compounds diminish the DNA damage produced by gamma-irradiation and also suggest that the mechanisms of radioprotection must be different. Thus, radioprotection mediated by DDC should involve free hydroxyl radical scavenging and a minor activity of exonuclease. The enhancement of phage induction in E. coli cells that DDC produces could be attributed to its quelant effect and this would not be not probably directly related to radioprotection. Adeturon, as thiols, may serve also as scavenging agent of free hydroxyl radicals, diminishing indirectly the DNA damage level. In addition, adeturon must interact with DNA in the same form that other aminothiol compounds do it. This interaction, mediated by amino groups of adeturon, may serve to concentrate these compounds near of the DNA damage site, increasing the potential for the thiol portion of the molecule to donate hydrogen, decreasing the damage level on DNA molecule. However, adeturon do not modify the exonuclease activity. Some topic about the possible clinical application of both compounds are discussed., (Copyright 1998 Elsevier Science B.V.)

The vasodilator nitric oxide (NO) is involved in the regulation of systemic blood pressure and local organ blood flow. Inhibitors of the constitutively expressed nitric oxide synthase in endothelial cells (eNOS), e.g., Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME), aggravated liver injury in a variety of models. On the other hand, inhibitors of the inducible NOS (iNOS), e.g., 2-aminoethyl-isothiourea (AET), were found to be beneficial during endotoxemia. The aim of this investigation was to study the effect of AET compared with L-NAME on liver microvascular blood flow and injury in more complex models with multiple insults, i.e., ischemia (20 min)-reperfusion (8 h) in combination with .5 mg/kg endotoxin (IRE). Male Fisher rats were treated with 10 mg/kg AET or L-NAME and subjected to IRE. At 8 h, liver injury (plasma ALT: 1320+/-164 U/L) was significantly increased in AET-treated (5,018+/-1,379 U/L) and L-NAME-treated groups (2,429+/-228 U/L). Each inhibitor attenuated microvascular blood flow (assessed by laser Doppler flowmetry) to a similar degree. In striking contrast, AET completely reversed the endotoxin-induced impairment of the microvascular blood flow and significantly protected against an endotoxin-induced liver injury (plasma ALT: 3,007+/-268 U/L (ET); 460+/-39 U/L (ET+AET)). Infusion of endothelin-1 reduced microvascular blood flow by 50-60% and caused liver injury. Our data demonstrated that an inhibitor of eNOS (L-NAME) has a consistent detrimental effect on liver injury during ischemia-reperfusion and endotoxemia mainly because it can cause additional ischemia by reducing the microvascular blood flow. However, selective inhibitors of iNOS (AET) can impair hepatic blood flow and aggravate the injury or improve blood flow and attenuate organ injury depending on the experimental model. These results suggest that iNOS inhibitors may not be universally beneficial and should be tested in a variety of experimental models of sepsis/endotoxemia before used in clinical settings.

The role of nitric oxide (NO) in hepatic oxygen transport is unclear. We investigated the effects of aminoethyl-isothiourea (AE-ITU), a selective inhibitor of iNOS activity, on liver blood flow and oxygen consumption (VO2H) in the pig. Endotoxin (lipopolysaccharide, LPS) was given intraportally (1.7 microg/kg/h), followed by AE-ITU (10 mg/kg) after 3 h (n = 7), LPS controls (n = 8) received LPS for 6 h. AE-ITU controls (n = 6) received saline/AE-ITU. LPS (treatment group) caused significant reductions at 3 h in cardiac output (CO) from 4.4 +/- .4 to 2.7 +/- .3 L/min, in hepatic artery flow (Q(HA)) from 266 +/- 53 to 127 +/- 19 mL/min, and in portal venous flow (Q(PV)) from 630 +/- 50 to 323 +/- 33 mL/min. Hepatic oxygen delivery (DO2H) was reduced from 93 +/- 11 to 38 +/- 5 mL/min (p < .05), while hepatic oxygen extraction ratio (ERO2H) increased, and VO2H was maintained. Similar changes were observed in LPS controls. AE-ITU caused no changes in saline controls. After injection of AE-ITU during LPS infusion, CO was unchanged, while Q(HA) increased gradually from 127 +/- 20 to 268 +/- 40 mL/min over 3 h (p < .05) and DO2H from 38 +/- 5 to 60 +/- 5 mL/in (p < .05). ERO2H increased from .54 +/- .04 to .69 +/- .03 in 30 min, while VO2H increased from 23 +/- 4 to 35 +/- 3 mL/in in 3 h (p < .05). Thus, AE-ITU restored hepatic arterial blood flow and increased hepatic oxygen consumption in pigs with endotoxemia.

Nowadays, labelled polyclonal and monoclonal antibodies are widely used for immunoscintigraphic diagnosis of different diseases. Technetium-99m is often considered to be the label of choice for radioimmunodiagnosis for reasons of cost, availability and imaging properties, in spite of its relatively short physical half-life (6.01 h). The existing labelling methods may be classified into two types: direct approaches, in which disulphide bridges within are reduced to generate endogenous sulfhydryl groups able to efficiently bind technetium due to their strong chelating capacity and indirect methods, in which an exogenous chelator is covalently attached to the protein to serve as the binding site. All these procedures have their advantages and drawbacks. There is no consensus among the authors about which of the methods is the best. The employed approach depends on the particular situation. The aim of the present work is to show an update about the available procedures for 99mTc-labelling of antibodies and its fragments.

Background/methods: The question whether nitric oxide protects or impairs organ perfusion during early endotoxemia has not been completely answered. To evaluate the regulative function of nitric oxide on organ microvascular perfusion and leukocyte accumulation during endotoxemia, we studied the influence of a non-selective nitric oxide inhibitor and a preferential inducible nitric oxide synthase inhibitor (respectively, N(G)-nitro-L-arginine methyl ester and aminoethyl-isothiourea) on liver microcirculation (intravital fluorescence microscopy) in a rat model., Results: Two hours after intraportal injection of lipopolysaccharide (5 mg/kg in 10 min) the rats were randomly treated and received a bolus dose of N(G)-nitro-L-arginine methyl ester (10 mg/kg, n = 7), aminoethyl-isothiourea (10 mg/kg, n = 6) or normal saline, (n = 7). After 1 h, N(G)-nitro-L-arginine methyl ester blockade yielded a higher rate of non-perfused sinusoids than normal saline (27 +/- 2% vs 19 +/- 5%, p < 0.05). LPS-induced leukocyte stagnation in sinusoids was further increased (p < 0.05) in all groups after 1 h treatment, but N(G)-nitro-L-arginine methyl ester clearly accentuated leukocyte accumulation in sinusoids as compared to normal saline (69 +/- 19% vs 16 +/- 4%, p < 0.05). Both modalities of nitric oxide blockade elicited a significant enhancement in the number of leukocytes adherent to the postsinusoidal venules in contrast to normal saline (N(G)-nitro-L-arginine methyl ester 48 +/- 17%, aminoethyl-isothiourea 33 +/- 9% vs normal saline 1 +/- 5%, p < 0.05)., Conclusions: We conclude that complete nitric oxide blockade aggravates lipopolysaccharide-induced hepatic microvascular perfusion failure and enhances leukocyte accumulation, in both sinusoids and post-sinusoidal venules. The preferential inducible nitric oxide synthase inhibitor aminoethyl-isothiourea has a moderate negative effect, favoring leukocyte adhesion in postsinusoidal venules, and its usefulness demands further research, especially concerning its late effects.

In an attempt to identify potent inhibitors of inducible (type II) NO synthase (iNOS) for use in cell culture systems, we found that two S-substituted isothioureas were very potent in cell culture but one such compound also interfered with the induction of NO synthase. S-Ethylisothiourea (EITU) and S-aminoethylisothiourea (AEITU) were found to be much more potent than NG-methylarginine, NG-nitroarginine methy lester, or aminoguanidine as inhibitors of NO production by cultured RAW 264.7 cell macrophages activated by lipopolysaccharide (LPS). The approximate EC50 values as inhibitors of NO production, assessed by 24-h accumulation in cell culture media, were 10 microM (EITU), 30 microM (AEITU), 300 microM (NG-methylarginine), and 1000 microM (aminoguanidine). EITU was found to inhibit NO production by activated macrophages without interfering with the induction of iNOS. More specifically, EITU failed to influence transcription of iNOS mRNA (Northern blot analysis), translation of iNOS protein (pulse experiments), or degradation of translated iNOS protein (pulse-chase experiments). In contrast, however, AEITU interfered markedly with the induction of iNOS by mechanisms attributed to inhibition of translation of iNOS mRNA into functional protein as well as acceleration of degradation of already translated iNOS protein. These observations indicate that AEITU should not be used in cell culture experiments where the intent is solely to assess the consequences of inhibition of iNOS catalytic activity.

Endotoxin has profound effects on nitric oxide (NO) production, and considerable controversies exist as to whether these alterations are beneficial or deleterious. Increased mortality has been reported from nonselective inhibition of NO synthase. Results from selective inhibition of the inducible isoform (iNOS) appear largely positive. In a model of rat endotoxemia we have compared the early effects on hepatic morphology and function of selective and nonselective NO inhibition. Two hours after endotoxin injection (5 mg/kg intraportally) the rats were treated with either the selective iNOS inhibitor aminoethyl isothiourea (AE-ITU, 10 mg/kg), the nonselective NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg), or normal saline. The animals were observed for another hour. Using an immunohistochemical method, induction of iNOS was demonstrated in various tissues in all slices examined. No unequivocal benefit from NO inhibition was noted. Electron microscopic examination revealed widespread alterations of liver morphology, without obvious differences between the groups. Liver function, as assessed by ketone body ratio, hepatic venous acid base values, and bile production, was generally more adversely affected after NO inhibition. Even with the iNOS selective inhibitor AE-ITU no benefit was noted. We conclude that during the early phases of endotoxemia therapeutic reduction of NO production has no positive effects on liver function or morphology.

The effect of inhibiting nitric oxide (NO) synthesis during sepsis was studied in a caecal perforation model on Wistar rats. This model induces severe abdominal sepsis with a 48-h mortality > 90% in untreated animals. The survival time in hours (median values with the 95% confidence intervals in parentheses) for the control group was 15 (11.5-27.0) h. Treatment with the inhibitors of NO synthesis, NG-monomethyl-L-arginine (LNMMA), 30 mg kg-1 body weight (BW) or S-(2-aminoethyl)-isothiourea (AET), 3 mg kg-1 BW, given either once or twice after sepsis induction did not affect survival in this model. Survival time when LNMMA was given once was 13 (11-22) h and when given twice it was 14 (8-41) h. The corresponding survival times were 9 (4-28) h and 11 (5-27) h for treatment with AET. In conclusion, this study demonstrates that survival in the present model of live multiplying bacterial sepsis is not affected by either LNMMA or AET. Testing the potential clinical effects of inhibition of NO synthesis during sepsis should not be confined to short-term studies of haemodynamic changes induced by lipopolysaccharide.

Objectives: To analyze the different immunohematologic studies required to identify anti-red cell antibodies directed against high incidence antigens and comment the best tranfusion management., Patients and Methods: Five patients with suspected anti-red cell alloantibodies directed against high frequency antigens are reported. After a positive antibody screening test (AST), an agglutination test with a commercial panel of 24 red cells was performed. Red cells were treated with proteolytic enzymes and AET to try to identify the circulating antibody. However, it was necessary to send the samples to reference laboratories for definitive identification. In order to evaluate the haemolytic potential of the antibody serum samples were treated with DTT and immunoglobulin subtype was studied with the capillary agglutination test. Finally, we analyze the half life of Cr51 labelled red cells. To obtain compatible blood for transfusion, autologous transfusion and cross-match with blood from direct relatives were performed., Results: AST was positive in every case. A decrease in the agglutination test was observed after ficin treatment in two patients, and an increase in the remaining. The treatment of red cells with ZZAP and AET resulted in a decrease of agglutination in three cases and an increase in the remaining two. Specificity of the antibodies was as follows: anti-Cellano (two cases), anti-Ku (one case) and anti-Yta (two cases). Anti-Kell antibodies were IgG1 and anti-Cartwright antibodies were IgG4. One patient was transfused with autologous blood alone, another patient received compatible blood from direct relatives. A third patient was transfused both with autologous and allogeneic compatible blood. The fourth patient did not need red cell transfusion and, finally the last patient had to be transfused with incompatible blood but no postransfusion haemolysis was observed., Conclusions: In patients with anti-red cell antibodies against high-frequency antigens, red blood cells treatment with proteolytic enzymes (ZZAP, ficin) and AET are useful techniques to approach to their identification. Beside this, the study of type and subtype of Ig are necessary to know the haemolytic activity of the antibody. Regarding the transfusional management, autologous transfusion, crossmatch with blood from direct relatives and cryopreservation of compatible blood are the most adequate attitudes to cover future needs.

In this study we compared the circulatory effects of the arginine analogue non-specific nitric oxide synthase (NOS) inhibitor N omega-nitro-L-arginine (NNA), and the specific inducible NOS (iNOS) inhibitor S-methylisothiourea (SMT) and S-(2-aminoethyl)-isothiourea (AEST) in a hyperdynamic endotoxemic dog model. Mean arterial pressure (MAP), cardiac output (CO), and myocardial contractility (MC) were measured. A hyperdynamic circulatory response was elicited with a 2-h infusion of a total dose of 5.3 micrograms/kg E. coli endotoxin (ETX). NOS inhibitory treatment (2 mg/kg) was administrated from the 45th min of endotoxemia. ETX induced a hyperdynamic circulatory response, and a significant myocardial depression. NNA induced a prolonged, SMT a transient increase in MC, both drugs elevated MAP, but decreased CO. AEST significantly prolonged the elevation in CO, but did not affect MAP. Selective inhibition of the iNOS may be a beneficial in sepsis.

Purpose: The protective effect of the sulphur-containing agents, MEA (cysteamine HCl), AET (2-aminoethyl-isothiouronium Br.HBr), and WR-2721 (S-2/3-aminopropylamino/ethylphosphorothioic acid) against induction of micronuclei in the erythropoietic system by X-rays was assessed., Methods: The mouse peripheral blood micronucleus assay was applied. The frequency of micronucleated polychromatic erythrocytes (MNPCE) in the peripheral blood of adult male Swiss mice was determined 24 h after MEA, AET, or WR-2721 treatment, at a dose of 200 mg/kg body weight, and whole-body X-ray exposure to a dose of 6 Gy., Results: After X-irradiation of mice, the number of MNPCE was distinctly increased. MEA, AET, or WR-2721 administration, 15 min prior to X-irradiation resulted in a reduction of the X-ray-induced elevation of the frequency of micronuclei. So, the radioprotection of the sulphur-containing drugs against the induction of MNPCE in the erythropoietic system by X-rays was shown., Conclusion: The mouse peripheral blood micronucleus test can be used to screen potential radioprotective agents.

The modulatory effect of AET (S-2-aminoethylisothio-uronium bromide hydrobromide) and WR-2721 (S-2-/3-ami-nopropylamino/ethylphosphorothioic acid) on the clastogenic activity of X-rays was assessed by the in vivo mouse micronucleus test. The frequency of micronucleated polychromatic erythrocytes (MNPCEs) in the peripheral blood of adult male Swiss mice exposed to 5 Gy X-rays alone, or treated with AET or WR-2721, at a does of 200 mg/kg body weight, 15 or 30 minutes prior to X-irradiation, respectively, was determined during a fifteen-day period. The number of micronuclei increased on day 1 post-irradiation in X-irradiated mice and declined thereafter with the frequency of MNPCEs remaining lower in the thiol pre-treated mice. A more effective protection against the clastogenic activity of X-rays in the erythropoietic system was observed after WR-2721 administration than AET application.

Calcium-loaded red blood cells (RBCs) previously have been shown to have an increased sensitivity to complement-mediated hemolysis and particularly to lysis mediated by the C5b-9 membrane attack complex (MAC) of complement. Because RBCs exposed to 2-aminoethylisothiouronium bromide (AET) also have been shown to be particularly sensitive to the MAC, a direct comparison of calcium-loaded and AET-treated RBCs was performed. Calcium-loaded and AET-treated RBCs shared a marked increase in sensitivity to lysis by the MAC in two different assays. However, measurements of C5b-7 and C9 binding suggested that different mechanisms were responsible. AET-treated RBCs showed an increase in C9 binding and an increased C9/C7 ratio consistent with functional loss of CD59/membrane inhibitor of reactive lysis (MIRL). In contrast, calcium-loaded RBCs had minimally increased C9 binding that resulted in C9/C7 ratios that were less than those for untreated RBCs, suggesting that CD59/MIRL inactivation had not occurred. When RBCs were incubated in acidified serum, AET-treated cells demonstrated a marked increase in C3b binding and hemolysis that was observed in neither control nor calcium-loaded RBCs. These results suggest that the underlying lesions responsible for an increase in susceptibility to complement-mediated hemolysis are different for calcium-loaded and AET-treated RBCs.

The effects of aminoethylisothiuronium bromide known as a radioprotector on the activity of human platelet soluble guanylate cyclase and on ADP-induced aggregation of human platelets have been studied. It has been shown that in Tris-buffer and at definite pH values aminoethylisothiuronium bromide is converted into mercaptoethylguanidine as a result of a transguanidine rearrangement. The latter contains in its molecule both guanidine and SH-groups which appear to be the donor and acceptor of nitric oxide, respectively. It was found that after its rearrangement into mercaptoethylguanidine, aminoethylisothiuronium bromide activates human platelet soluble guanylate cyclase, inhibits ADP-induced aggregation of human platelets and accelerates their spontaneous disaggregation. The stimulating effect of aminoethylisothiuronium bromide depends on the effectiveness of its transguanidine rearrangement into mercaptoethylguanidine. A molecular mechanism of the hypotensive side effect of aminoethylisothiuronium bromide is proposed.

Effects of aminoethylisothiuronium bromide (AET), known as radioprotector, on human platelet soluble guanylate cyclase and on ADP-induced human platelets aggregation were studied. It was shown that AET - in Tris buffer and at certain pH values - is converted, via transguanidine rearrangement, to mercaptoethylguanidine. The latter contains in its molecule both the guanidine and SH groups which act as donor and acceptor of nitric oxide (NO), respectively. It was demonstrated that AET, after its rearrangement to mercaptoethylguanidine, is able to activate human platelet soluble guanylate cyclase, as well as to inhibit ADP-induced human stimulatory effect of AET is dependent on the effectiveness of its transguanidine rearrangement to mercaptoethylguanidine. The molecular mechanism of the hypotensive by - effect of AET is proposed.

Unlabelled: Radionuclides employed in diagnostic and therapeutic nuclear medicine impart radiation energy to tissue over an extended period of time, which depends on the physical half-life and the biological properties of the radiochemical employed. It is therefore important to examine the capacity of chemical radioprotectors to mitigate damage caused by chronic irradiation by incorporated radionuclides., Methods: Spermatogenesis in mouse testis is used as the experimental model, and spermatogonial cell survival as measured by testicular spermhead count is the biological end point. The capacity of S-(2-aminoethyl)isothiouronium bromide hydrobromide (AET) to mitigate radiation damage caused by chronic irradiation by the radiochemicals 125IUdR, H125IPDM and 210Po-citrate, is investigated., Results: The radioprotection provided by AET is substantial and similar for both of the radioiodinated compounds with dose modification factors (DMF) of 4.0 +/- 1.2 for 125IUdR and 3.4 +/- 0.4 for H125IPDM. In contrast, the damage caused by 210Po alpha particles is protected against to a lesser degree (DMF = 2.4 +/- 0.5)., Conclusion: The present radioprotection data for AET, in conjunction with our earlier findings for the chemical protectors cysteamine and vitamin C in the same experimental model, suggest that such compounds may be clinically useful as mitigating agents against biological damage caused by incorporated radionuclides. The observed DMFs for AET also support our earlier premise that the mechanism by which DNA-incorporated Auger emitters impart biological damage is primarily radical mediated, and hence indirect in nature.

Aminothiol radioprotectors (MEA, AET, WR-1065) were found to be inhibitors of platelet aggregation. The aim of this study has been to clarify the potential involvement of the adenylate cyclase-cAMP system in this effect. In vitro aminothiol compounds (> 1 mmol/l) inhibited platelet function as well as AC-activity, i.e. significantly reduced their cAMP content. In less than 1 mmol/l concentration WR-1065 elevated the cAMP content of PGI2-stimulated platelets, while basal cAMP level mainly remained unchanged. On the effect of WR-1065 the level of non-protein thiols (NP-SH) markedly elevated in the platelets, while their protein-bound thiol content (PB-SH) either decreased or remained at control level. The phosphorothioate WR-2721 was effective only after in vitro or in vivo dephosphorylation. Although the amount of NP-SH slightly increased, the PB-SH content decreased in the platelets isolated after intravenous administration of WR-2721. Moreover, platelet-poor plasma samples of WR-2721 treated animals also inhibited both the AC-activity and function of control platelets, too. Based on these results it is suggested that aminothiol radioprotectors non-specifically inhibit platelet functions forming mixed disulfides with endogenous, mostly protein-bound thiols. The slight elevation of AC-activity caused by low doses of WR-1065 also suggest that the AC-complex consists of unevenly sensitive subunits (G-proteins, catalytic subunit).

Radioprotective effectiveness of a combination of HT and AET was evaluated by radiation induced micronucleus screening in mouse bone marrow. Swiss albino mice were injected with 2-Aminoethylisothiouronium bromide hydrobromide (AET)+5-hydroxy-L-tryptophan (HT) 30 minutes prior to a single whole body exposure of 8 and 12 Gy Co60 gamma rays. Animals were sacrificed at different intervals viz., 24 hours, 40 hours, 72 hours and 7 days after irradiation. Slides of femur marrow were screened thoroughly for cells with micronuclei. Marked decreases in the frequency of micronuclei in irradiated mice receiving radioprotector suggested that the combination has rendered protection to bone marrow cells against 8 Gy and 12 Gy whole body gamma irradiation. Micronucleated cells count is not altered by administration of the combination alone.

Lymphocytes treated with a 6% and 4% AET solution after 60 or 120 minutes at 20 degrees or 37 degrees C display polyreactivity. Polyreactivity does not develop after short-term treatment (1-10 mins.). After treatment of lymphocytes with a 3% or 2% AET solution for a period of 20 or 30 mins. at 20 degrees or 37 degrees C the specific expresivity of HLA--A, B antigens rises; they are manifested, contrary to untreated lymphocytes, also by weak HLA sera. Addition of AET to the mixture of lymphocytes--HLA serum causes in the first stage of the microlymphocytotoxic test negativity of the test due to a block of the haemolytic activity of rabbit complement.

Different patterns of temporary changes were observed in the acid phosphatase/E.C. 3.1.3.2./activity in the spleen, liver, and kidneys expressed in nkat per 1 mg of protein, 1 g of fresh tissue, and per whole organ weight in mice exposed to 60Co gamma-rays only and those irradiated after AET (2-aminoethylisothiouronium Br.HBr) or MEA (cysteamine HCl) treatment. The extent and timing of the alterations in enzyme activity was dependent on the particular organ chosen and the thiol agent applied. AET and MEA administered before irradiation modified the effects of exposure of mice to gamma-rays on the acid phosphatase activity in the organs studied.

Differential radioprotection between normal tissues and carcinoma was observed in C3H/J mice treated with a combination of 5-hydroxy L-tryptophan (5-HTP, 100 mg/kg) and 2-aminoethylisothiuronium bromide hydrobromide (AET, 20 mg/kg). Protection to normal tissues was judged by LD50(30) and by radiation induced damage to bone marrow(BM) using clonogenic ability of blood forming stem cells (10 day CFUs) as the criteria. Pretreatment with 5-HTP + AET combination 30 min before whole body gamma radiation (WBGR) enhanced the recoveries of the number of blood forming stem cells in BM of irradiated mice after 0, 7th and 10th day of irradiation. LD50(30) for C3H/J mice was 7.3 Gy and the dose modifying factor (DMF) of 5-HTP + AET combination was 1.76. On the contrary, pretreatment with this combination did not protect the mammary carcinoma transplanted in C3H/J mice, when exposed to 80 Gy soft X-rays.

Sperm abnormalities and fall in total sperm count following different doses (4 Gy, 5 Gy and 6 Gy) of whole body gamma irradiation (WBGR) were studied in adult male Swiss strain A mice. The protecting ability of a combination of 5-hydroxy-L-tryptophan (5-HTP, 100 mg/kg) and 2-aminoethyl isothiuronium bromide hydrobromide (AET, 20 mg/kg) was also investigated. Pretreatment with a 5-HTP+AET formulation i.p., 30 min before irradiation modified the fall in sperm counts significantly. Exposures to 4 Gy, 5 Gy and 6 Gy WBGR caused marked increase of sperm abnormalities which could be significantly reduced by pretreatment with 5-HTP-AET. WBGR with 4 Gy, 5 Gy and 6 Gy produced a short period of sterility associated with oligospermia but these abnormalities were corrected by pretreatment with 5-HTP+AET. This finding was supported by breeding experiments in pretreated adult male Sprague-Dawley rats which showed delivery of normal offsprings in drug-protected irradiated groups in contrast to irradiated controls.

The results showed that marrow stromal cells include fibroblasts, reticular cells, macrophages and adipocytes. The capability of the adherent layer derived from marrow cells of 2 mouse femurs to support hematopoietic stem cells was stronger than those of layers derived from 0.5 or 1 mouse femurs. The radiosensitivity of bone marrow stromal cells was lower than that of hematopoietic stem cells. The radioprotective effect of AET and PLP (polysaccharide of Lobaria pulmonaria Hoffm) on the bone marrow stromal cells and their capability to support hematopoietic stem cells was clearly demonstrated.

These experiments continued our line of inquiry into low-toxicity high-effectiveness radiomodifying agents obtained by binding radioprotective substances to biogenic and biologically active components or antidotes. Testes for toxicity and radioprotective effects were three preparations combining within one molecule AET and an adenyl nucleotide (AMP, ADP, or ATP). Ionic-bond formation was shown to have advantages over concomitant administration of AET and adenosine phosphoric acids as mixtures. The evidence obtained supports the rational foundation of the concept being developed by our laboratory.

To investigate the mechanism by which treatment of normal human erythrocytes with the sulfhydryl reagent 2-aminoethylisothiouronium bromide (AET) induces susceptibility to complement mediated lysis, the effects of AET on the structural and functional integrity of decay accelerating factor (DAF), membrane inhibitor of reactive lysis (MIRL), and complement receptor type 1 (CR1) were examined. Following treatment with AET, erythrocyte MIRL and CR1 were no longer recognized in situ by antibodies, and antibody binding to DAF was diminished by approximately 50%. These studies indicated that the structural integrity of the three complement regulatory proteins was either partially (DAF) or completely (MIRL and CR1) disrupted by AET. Subsequent experiments showed that functional inactivation paralleled the structural disruption. Treatment of normal erythrocytes with AET induced susceptibility to cobra venom factor-initiated hemolysis, indicating that the functional activity of MIRL had been destroyed. The capacity of erythrocyte CR1 to serve as a cofactor for factor I-mediated cleavage of iC3b to C3c and C3dg was lost following treatment with AET. C3 convertase activity increase markedly following treatment of erythrocytes with AET, but convertase activity on AET cells was approximately 50% less than that observed when DAF function on normal cells was completely inhibited by antibody. Susceptibility of AET cells to acidified serum lysis was shown to be due primarily to inactivation of MIRL. Unexpectedly, in acidified serum the activity of the amplification C3 convertase of the APC was found to be controlled by MIRL as well as by DAF. These studies show that AET induces susceptibility to complement-mediated lysis by disrupting the structural and functional integrity of membrane constituents that regulate the activity of both the C3 convertases and the membrane attack complex of complement.

A study of C4 bound to human erythrocytes in vitro and in vivo has been made by immunoblotting with mouse monoclonal anti-C4c and anti-C4d and human polyclonal anti-C4d (Rodgers and Chido) following SDS-PAGE. Multi-banded patterns differentiated between C4A and C4B isotypes. Treatment of EC4b with trypsin eliminated immunoblotting but not agglutination reactions. Serum inactivation (factor I) of EC4b resulted in banding patterns similar to those obtained from patients' EC4d. Treatment of EC4b membranes with NH2OH affected many of the bands, two were lost, one was markedly reduced and others had altered SDS-PAGE mobility. Interpretation of the bands has been made in terms of C4-acceptor complexes and inactivation fragments of C4. A distinct difference in the banding of C4A and C4B isotypes has been detected.

The adult male Swiss mice were either whole-body gamma-irradiated with a single dose of 10 Gy from 60Co source, always at 19.00 or, 15 minutes before irradiation injected intraperitoneally with AET (2-aminoethylisothiouronium Br. HBr), or MEA (cysteamine HCl), in a dose of 400 mg/kg body weight. The measurements of the protein level in crude homogenates of liver were done in four-hour internals during a 24-hour period, starting at 20.00. The protein concentration in liver was calculated per 1 g of fresh tissue and the whole organ weight. The body and liver weight was also studied. There were no fluctuations in the liver weight and concentration of protein in the control and irradiated only mice. Temporary changes in the liver weight and level of protein expressed in mg per 1 g of fresh tissue and the whole organ weight could be found in the group of males treated with AET, and daily changes in the liver weight and concentration of protein related to mg per 1 g of fresh tissue, in the group of male mice injected with MEA prior to irradiation, could be recorded. Differences in the liver weight at 20.00, 24.00 and 04.00, as well as in the protein level expressed in mg per 1 g fresh tissue at 04.00, 12.00, 16.00, and the whole liver weight at 24.00, 04.00, and 16.00, of between the particular groups of mice, were observed. There were no temporary changes in the body weight in any of the groups and there were no differences in this value between the groups of mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Temporal changes of acid phosphatase (E.C. 3.1.3.2) and arylsulphatase (E.C. 3.1.6.1) activities in testes of adult Swiss mice after AET (2-amino-ethylisothiouronium Br. HBr) or MEA (cysteamine HCl) treatment, were studied. The animals were injected intraperitoneally with the S-containing substances in a single dose of 400 mg/kg body weight. The enzyme activities in crude organ homogenates were assessed every four hours during a 24-hour period. Administration of the aminothiol agents to mouse organism caused greater changes in the acid phosphatase activity than in the arylsulphatase activity, and the two chemical compounds AET and MEA given, influenced the enzyme activities in testes in a different way. Treatment of mice with AET resulted in a decrease of the acid phosphatase activity related to 1 g of fresh tissue at 16.00 and the whole organ weight at 24.00 and 16.00 as well as in a decrease of the arylsulphatase activity expressed per the whole weight of testes at 08.00. After MEA injection, the acid phosphatase activity related to 1 mg of protein, 1 g of fresh tissue and the whole organ weight was decreased at 20.00(1), and the enzyme activity expresse per 1 mg of protein and 1 g of fresh tissue was increased at 24.00, but the arylsulphatase activity related to both 1 mg of protein at 08.00, 12.00 and to the whole weight of testes at 08.00, was reduced.

A statistically significant decrease was found in the glycogen content expressed in mg per 1 g of both liver in groups of mice killed in the 60th and 90th minute, and skeletal muscles in those killed in the 30th, 60th, and 90th minute after MEA injection, as well as a statistically significantly reduced blood glucose concentration in a group of mice killed in the 30th minute after AET treatment.

Unirradiated mice were given i.p. a mixture of mexamine (20 mg/kg body weight) and AET (150 mg/kg body weight) or mexamine (20 mg/kg body weight) and cystamine (150 mg/kg body weight). Acute effects of these mixtures of radioprotective agents on the ultrastructure of basophilic and polychromatic bone marrow erythroblast mitochondria were studied. One hour after injecting mice with above-mentioned mixtures, statistically significant differences in the size of mitochondria were found between control mice and mice given a mixture of mexamine and cystamine. The differences in the number of dilated cristae per micron 2 of the mitochondrion area between control mice and both experimental groups given mixtures of radioprotective agents were also statistically significant. Thus it was proved that also mixtures of radioprotectors induced acute ultrastructural changes of mitochondria which were so far described only after treating mice with only one radioprotective agent.

The adult male Swiss mice were either whole-body 60Co gamma-irradiated with a single dose of 10 Gy at 1900, or treated intraperitoneally with AET (2-aminoethylisothiouronium bromide hydrobromide) or MEA (cysteamine hydrochloride), in a dose of 400 mg/kg body weight, 15 minutes before irradiation. The measurements of the arylsulphatase (E.C. 3.1.6.1) activity in crude homogenate of liver were done every four hours during 24-hour period, starting at 2000. The enzyme activity towards the artificial substrate p-nitrocatechol sulphate was related to 1 mg of protein, 1 g of fresh tissue and to the whole organ weight. No fluctuations in the activity of arylsulphatase in the control mice were found. There were temporary changes in the enzyme activity expressed in nkat per 1 mg of protein and 1 g of fresh tissue in the irradiated only males, in that related to 1 g of fresh tissue and the whole organ weight in mice injected with AET before irradiation, as well as that calculated in nkat per 1 mg of protein in animals pre-treated with MEA to exposure of gamma rays. Between the particular groups of males, differences in the arylsulphatase activity related to 1 g of fresh tissue and the whole liver weight at 0400 were only observed. 60Co gamma-irradiation influenced the activity of arylsulphatase in liver. AET and MEA modified the effect of exposure of mice to gamma rays on the enzyme activity in a different way.

HLA class I antigens (Bg) on red cells (RBCs) are expressed by some normal donors and by many patients with systemic lupus erythematosus (SLE). To identify the membrane components previously detected by hemagglutination with HLA class I-specific monoclonal antibodies (MoAbs), RBC membrane preparations were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with the HLA class I MoAbs. Two components were obtained that reacted with the MoAbs: a heavy chain of 45 kDa and a light chain termed beta2-microglobulin (beta2-M) of 11 kDa. The effect of chloroquine and acid elution in stripping HLA antigens is shown to be due to the removal of beta2-M, as only that component was detected in eluates from reactive RBCs. Neither antibody elution method affected the heavy chain expression assessed by immunoblotting. It is concluded that HLA class I antigens on RBCs are integral membrane components of the type normally found and wisely distributed on many nucleated cells. Platelets, which have stronger HLA class I antigen expression, were also studied, and their membrane preparations yielded heavy chain and beta2-M molecules; the effect of chloroquine treatment was harder to assess than that of acid elution, owing to the sensitivity with which both components are detected in immunoblotting. In eluates obtained from acid treatment only beta2-M is detected.

The changes in the amounts of blood and tissular sulfhydryl groups and lipid peroxides were investigated in lethally gamma-irradiated rats treated with cystamine, AET (radioprotectors) or alpha-methyl-dopa (a radiosensitizer). The results of experiments revealed the following findings: 1. The ionizing radiation generally causes increases in the levels of tissular sulfhydryl groups and lipid peroxides (excepting the hepatic levels of these compounds). 2. AET and, especially, cystamine are able to reverse the radiation-induced changes in the tissular amounts of sulfhydryl groups and lipid peroxides; there are close relationships between this effect (including its intensity and extension) and the radioprotective action of these drugs. 3. In spite of its radiosensitizing action, alpha-methyl-dopa induces approximately similar changes in the tissular amounts of sulfhydryl groups and lipid peroxides, suggesting that biochemical processes other than an excessive yield of free radicals are also involved in radiosensitivity.