Effect of gamma radiation on the immunobiological and immunochemical properties of cholera exotoxin. III. The serological activity and immunochemical properties of irradiated unpurified toxin

The immunochemical properties and serological activity of irradiated preparations of crude cholera exotoxin have been studied. This study has revealed that with the increase of the dose of ionizing radiation changes occur in the physico-chemical properties of the preparations of the toxin, which leads to an increase in the electrophoretic motility of the protein components of the toxin, to the aggregation and polymerization of individual fragments. The preparations of antigen exotoxins have been shown to retain their serological activity within the range of radiation doses under study (10-350 kGy).     

Stimulating action of Brucella melitensis lipopolysaccharide on hemopoiesis in mice

The bacterial mass, brucellar protective antigen and lipopolysaccharide (LPS) obtained from B. melitensis stimulated the formation of endogenous colonies in the spleen of mice belonging to different strains, subjected to irradiation in sublethal doses. The maximum stimulating effect was observed when the antigens were introduced 24 hours prior to irradiation. LPS introduced in the optimal dose induced an increase in the number of hemopoietic stem cells (HSC) in the s-phase of the cell cycle, thus stimulating the postirradiation survival of mice irradiated in a lethal dose. 24 hours after the injection of LPS the total number of HSC in the spleen increased 1.5 times. These data indicate that LPS has a stimulating effect on hemopoiesis in mice. The effect rendered by LPS is seemingly linked with an increase in the proliferation of HSC and, to a lesser extent, depends on changes in the migration of HSC.     

Immunochemical characteristics and serologic properties of lipopolysaccharides isolated from various Brucella species using various extraction methods

The results of the comparative analysis of LPS isolated by different methods of extraction from the cultures of several Brucella species differing in their virulence are presented. Purified LPS preparations have been obtained from Brucella virulent and vaccine strains by using such methods as water-phenol extraction, Boivin's method, mild alkaline hydrolysis of the antigen according to Boivin's procedure. The presence of certain relationship between the method used for the extraction of Brucella LPS to be compared and their chemical composition, immunological characteristics and serological activity has been established. As shown in this investigation, in the process of the preparation of a highly sensitive diagnosticum for the passive hemagglutination test the use of LPS obtained from Brucella virulent strains, but not from the vaccine strain, by the method of mild alkaline hydrolysis according to Boivin's procedure is expedient. The data presented in this work indicate that the soluble complex of lipid A obtained from Brucella LPS has been found to possess serological activity. The results of the study of the serological properties of lipid A indicate that the lipid component may also play a certain role in the manifestation of the serological activity of Brucella LPS.     

Immunogenicity of the surface polysaccharide of serotype A meningococci possessing different physico-chemical properties

The influence of changes in the physico-chemical parameters of serogroup A meningococcal polysaccharide on its immunogenicity for mice was studied by means of passive local hemolysis in gel and the passive hemagglutination test. The polysaccharide was depolymerized by heating at 100 degrees C for 5, 30 and 120 minutes; during this process the progressing decrease of the molecular weight and the content of O-acetyl groups in the preparation could be observed. Mice showed high sensitivity to changes in the above-mentioned physico-chemical parameters, which was manifested by a sharp drop in the intensity of the immune response of the animals to the heated samples of the antigen. The role of the parameters under study, i. e. the molecular weight of the antigen and the presence of O-acetyl groups, in the manifestations of the immunogenicity of polysaccharide A is discussed.     

Modifying properties of 2,5-diphenyloxazole during low-dose X-ray exposure of mice

The ability of 2,5-diphenyloxazole (DPO) to modify biological consequences of the X-rays irradiation of mice was studied with a dose of 16 cGy at the administration of the agent in a wide range of concentrations before or after irradiation was studied. It was shown that the administration of the agent in doses 9.9 x 10(-3)-9.8 mg/kg 35-60 min before irradiation causes a reliable decrease in the spleen mass within 1 month after the action; for the dose 1 mg/kg, it causes the tendency to decrease of the content of lipid peroxidation (LPO) products; the dose 9.8 mg/kg causes a decrease in the cell-free DNA amount in blood plasma of mice. The administration of DPO before irradiation causes changes in the scale and direction of the correlation between the DNA and LPO products contents in blood plasma of irradiated mice compared with the control. The administration of DPO 15-60 min after irradiation do not cause any reliable changes in the investigated parameters. The aviability of the study of the radioprotective properties of the DPO derivatives as agents with a nontraditional character of action is supposed.     

Antigenic properties of LPS extracted from Bacteroides fragilis enterotoxin producing strains

Antigenic properties of enterotoxigenic Bacteroides fragilis (ETBF) strains isolated in Poland were compared with reference strains. The agglutination and passive hemagglutination, SDS-PAGE analysis and immunoblotting tests as well as analyses of sugars and fatty acids were performed with lipopolysaccharide (LPS) preparations obtained from water-phase of phenol-water extracts. Some differences in serological reactivity between ETBF antigens were observed. The antigen of the NTBF (nonenterotoxigenic) reference strain IPL E-323 expressed weak cross-reactivity with sera against whole cells of ETBF strains in serological tests. There were some differences observed between ETBF and NTBF strains in fatty acids and sugar composition. The LPS preparations probably possess a common core structure and the O-specific polysaccharides of variable chain length.     

Effect of immunization with lipid associated polysaccharide antigen and anti CD-2 antibodies on class II MHC expression and cellular immune response in BALB/C mice infected with Leishmania donovani

In a bid to characterize the antigens and immunization mechanisms which may be used to produce a protective response against L. donovani, role of lipid associated polysaccharide (LPS) antigen and whole antigen was evaluated. BALB/C mice were immunized with whole or LPS antigen in combination with one of three putative adjuvents (anti CD-2 antibody/FIA/0.85% Saline). LPS antigen emulsified in anti CD-2 antibody was found to induce significant antibodies in mice on day 28 against challenge with lethal dose of L. donovani. Immunoprophylactic properties of LPS and whole antigen was investigated on day 40 through cytokine elicitation (IL-2), MIF) in culture supernatants of spleen cells, but before that MHC-II expressed on macrophage was studied. The LPS antigen in combination with anti CD-2 antibody was found to be most immuno-reactive inducing higher MHC-II expression on macrophages which was associated with substantial rise in the level of MIF and IL-2. It coincided with decline in antibody titre in 100% mice immunized with LPS antigen while Leishmania injected as whole antigen failed to induce specific macrophage and T-cell response with all the above formulations. We surmise from our data that lipid associated polysaccharide antigen linked to anti CD-2 antibody has potential for eliciting protective immunity against Leishmania.     

Lipopolysaccharide-Induced Striatal Nitrosative Stress and Impaired Social Recognition Memory Are Not Magnified by Paraquat Coexposure

Systemic inflammation triggered by lipopolysaccharide (LPS) administration disrupts blood–brain barrier (BBB) homeostasis in animal models. This event leads to increased susceptibility of several encephalic structures to potential neurotoxicants present in the bloodstream. In this study, we investigated the effects of alternate intraperitoneal injections of LPS on BBB permeability, social recognition memory and biochemical parameters in the striatum 24 h and 60 days after treatments. In addition, we investigated whether the exposure to a moderate neurotoxic dose of the herbicide paraquat could potentiate LPS-induced neurotoxicity. LPS administration caused a transient disruption of BBB integrity, evidenced by increased levels of exogenously administered sodium fluorescein in the striatum. Also, LPS exposure caused delayed impairment in social recognition memory (evaluated at day 38 after treatments) and increase in the striatal levels of 3-nitrotyrosine. These events were observed in the absence of significant changes in motor coordination and in the levels of tyrosine hydroxylase (TH) in the striatum and substantia nigra. PQ exposure, which caused a long-lasting decrease of striatal mitochondrial complex I activity, did not modify LPS-induced behavioral and striatal biochemical changes. The results indicate that systemic administration of LPS causes delayed social recognition memory deficit and striatal nitrosative stress in adult mice and that the coexposure to a moderately toxic dose of PQ did not magnify these events. In addition, PQ-induced inhibition of striatal mitochondrial complex I was also not magnified by LPS exposure, indicating the absence of synergic neurotoxic effects of LPS and PQ in this experimental model.

     

N-acetylcysteine and glutathione as inhibitors of tumor necrosis factor production.

TNF is a major mediator in the pathogenesis of endotoxic shock, and its inhibition has a protective effect in various animal models of sepsis or endotoxin (lipopolysaccharide, LPS) toxicity. LPS treatment also induces an oxidative damage mediated by increased production of reactive oxygen intermediates. N-Acetylcysteine (NAC) is an antioxidant and a precursor of the synthesis of glutathione (GSH) and was reported to protect against LPS toxicity and LPS-induced pulmonary edema. In this study we investigated the effect of NAC on TNF production and LPS lethality in mice. The results indicated that oral administration of NAC protects against LPS toxicity and inhibits the increase in serum TNF levels in LPS-treated mice. The inhibition was not confined to the released form of TNF, since NAC also inhibited LPS-induced spleen-associated TNF. On the other hand, the inhibitor of GSH synthesis, DL-buthionine-(SR)-sulfoximine (BSO), had the opposite effect of potentiating LPS-induced TNF production, and this was associated with a decrease in liver GSH levels. Repletion of liver GSH with NAC reversed this effect. NAC was also active in inhibiting TNF production and hepatotoxicity in mice treated with LPS in association with a sensitizing dose of Actinomycin D. These data indicate that GSH can be an endogenous modulator of TNF production in vivo. On the other hand, NAC pretreatment did not inhibit other effects of LPS, particularly induction of serum IL-6, spleen IL-1 alpha, and corticosterone, in the same experimental model, suggesting that the observed effect could be specific for TNF.     

Structural elucidation of the Brucella melitensis M antigen by high-resolution NMR at 500 MHz

The Brucella M antigen from the species type strain Brucella melitensis 16M has been identified as a component of the cell wall lipopolysaccharide (LPS). O polysaccharide liberated from this LPS by mild acid hydrolysis exhibited M activity in serological tests and was shown to be a homopolymer of 4-formamido-4,6-dideoxy-alpha-D-mannopyranosyl residues arranged in an oligosaccharide repeating unit as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the native lipopolysaccharide. Structural analysis of the O polysaccharide by NMR methods was difficult due to apparent microheterogeneity of the repeating unit, which was in fact caused by the presence of rotational isomers of the N-formyl moiety. This problem was resolved by chemical modification of the polysaccharide to its amino and N-acetyl derivatives, the 500-MHz 1H and 125-MHz 13C NMR spectra of which could be analyzed in terms of a unique structure through application of pH-dependent beta-shifts and two-dimensional techniques that included COSY, relayed COSY, and NOESY experiments together with heteronuclear C/H shift correlation spectroscopy. On the basis of these experiments and supported by methylation and periodate oxidation data, the structure of the M polysaccharide was determined as a linear polymer of unbranched pentasaccharide repeating units consisting of four 1,2-linked and one 1,3-linked 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl residues. The marked structural similarity of the M antigen and the A antigen, which is known to be a 1,2-linked homopolysaccharide of 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl units, accounts for cross-serological reactions of the two and the long-standing confusion surrounding the nature of their antigenic determinants. Structural and serological considerations in conjuction with the sodium dodecyl sulfate banding pattern of Brucella A LPS suggest that its biosynthesis differs appreciably from that of the M antigen, which appears to be synthesized by regulated assembly of preformed oligosaccharide repeating units. Temperature, lysogenic phage may be responsible for such biosynthetic and structural variations.     

The modifying effect of beta-carotene on gamma radiation-induced elevation of oxidative reactions and genotoxicity in male rats

The aim of the present work was to evaluate the modulatory role of beta-carotene on the radiation-induced changes in certain biochemical and cytogenetic parameters. beta-Carotene was given by gavage at a dose of 5 mg/kg body weight for 7 consecutive days before whole body gamma irradiation with 7 Gy (single dose). The levels of beta-carotene in plasma, thiobarbituric acid-reactive substances (TBARS) in plasma and liver, the activities of superoxide dismutase (SOD) and catalase in blood and liver were the selected parameters. Furthermore, the frequency of micronuclei (MN) of polychromatic erythrocytes (PCEs), normochromatic erythrocytes (NCEs), the ratio of PCEs/NCEs and the mitotic index (MI) of bone marrow cells were also evaluated. The biochemical and cytogenetic determinations were carried out 1, 24, and 72 h after radiation exposure.The results obtained revealed that administration of beta-carotene pre-irradiation significantly inhibited the decrease in plasma beta-carotene, significantly reduced the levels of TBARS in plasma and liver. Significant protection of the radiation-induced changes in the activities of SOD and catalase was also recorded in the blood and liver of beta-carotene-treated and -irradiated rats. beta-Carotene resulted in significant inhibition in the frequency of radiation-induced MN, as well as in the ratio of PCEs/NCEs and the MI of bone marrow cells. These results suggest that beta-carotene as a natural product with its antioxidant capacity and capability of quenching singlet oxygen, could play a modulatory role against the cellular damage affected by free radicals induced by whole body irradiation.     

Action of x-ray irradiation on the nuclear envelope of rat liver cells]

X-irradiation of isolated nuclear envelopes (NE) has revealed their high radiosensitivity, while irradiation of isolated intact nuclei in vitro, in the doses up to 5000 r 18--20 hours after partial hepatectomy, produced no morphological changes in NE. The damaging effect of irradiation on both nuclei and mitochondria (Mt) was revealed only with a decrease in cytochrome-c-oxidase (CO) activity in parallel with an increase in the radiation dose. One hour after the whole body irradiation of rats in the beginning of S-period, the damaging effect was recorded in both NE and Mt at the doses of 50 and 150 t, and was enhanced with the increase of irradiation dose. Morphological changes were observed mostly in the outer nuclear membrane, which lost its distinct outline and disappeared from some nuclear regions. Lethal radiation doses produced a decrease in the number of pore complexes (PC) with their evident segregation from the membranes. After irradiation in a dose of 1200 r, only the residue or "ghosts" of the PCs remained. After irradiation in doses up to 400 r, the CO-activity recovered during the first hour in Mt and during first two hours in the nuclei.     

Serological cross-reaction between intact and chemically modified lipopolysaccharides of O1 Vibrio cholerae Inaba and non-O1 V. cholerae bio-serogroup Hakata.

Serological cross-reaction of intact as well as chemically modified LPS from O1 Vibrio cholerae 569B (Inaba) with non-O1 V. cholerae Hakata LPS, which contain alpha(1-->2)-linked N-acetyl perosamine-homopolymer constituting their O polysaccharide chain, was studied by passive hemolysis test by using their LPS as antigen for sensitizing sheep red blood cells (SRBC). The N-deacylation of the alpha(1-->2)-linked linear 3-deoxy-tetronyl perosamine-homopolymer constituting the O polysaccharide chain in 569B LPS resulted in virtual elimination of their serological reactivity with both homologous Inaba and heterologous Hakata antisera. Furthermore, when the resultant NH2 groups of the N-deacylated perosamine-homopolymers in 569B LPS were N-acylated with acetyl, propionyl or butanoyl groups, they markedly recovered the serological reactivity to a marked extent, in particular, their pronounced cross-serological reactivity with heterologous Hakata antiserum. These results are believed to be compatible with the interpretation that the Inaba antigen factor C possessed by the two bacteria studied is related to the common occurrence of the N-acyl groups, regardless of what the acyl groups are, residing in the perosamine residues of the perosamine-homopolymers constituting the O polysaccharide chain of their LPS.     

Changes in functional activity of the synthetic apparatus of rat thymocytes under acute and chronic gamma-irradiation

The changes in functional activity of rat thymocyte synthetic apparatus (synthetic activity) under acute (7.5 Gy) and continuous (dose rates 14.4 and 0.43 cGy/day) gamma-irradiation were studied by the fluorescent microspectral analysis. It has been shown that after the acute irradiation the changes in synthetic activity occurred in three main stages. The stages reflect the depression and activation of synthetic processes that is due to interphase and reproductive cell death and urgent recovery of thymus cellularity and secondary repopulating. Under continuous irradiation with a dose rate 14.4 cGy/day in long-term period both the decrease of thymocyte synthetic activity (in most animals) and activation (in the animals with pronounced symptoms of radiation damage) were observed. This reflects the depression processes in immune system and augmentation of immunoreactivity due to mass antigen influence of transformed cells and infectious agents on thymocytes. Under low dose ionizing irradiation (dose rate 0.43 cGy/day) the undulating changes in synthetic processes in thymus cells were observed. This depends on the recurrence of depression and recovery processes in the blood-forming tissue.     

Influence of a specific histone kinase on the physico-chemical properties of chromatin In situ

The influence of a specific histone kinase, phosphorylating lysine-rich histone F₁, F_2a2, F_2b, on the physico-chemical properties of the chromatin in the whole undestroyed fixed cell, has been investigated.It was found that the exogenous histone kinase penetrates into the nuclei of the undestroyed fixed cells and into the isolated unfixed nuclei and changes the physico-chemical properties of the chromatin there, bringing about an increase in binding of a basic dye acridine orange and a decrease in its stability to heat.     

Bimodal changes in germinability of pea seeds under the influence of low doses of gamma-radiation

Pea seeds (cv. 'Nemchinovskii-85', harvest of 2002, 80%-germination percentage) were exposed to gamma-radiation with doses ranging from 19 cGy to 100 Gy. One week after the irradiation with doses of 19 cGy and 3 Gy. the germination percentage decreased to 58 and 45%, respectively; at doses of 7 and 10 Gy it was 73 and 70% respectively. At greater doses (25, 50, and 100 Gy), germination percentage decreased in proportion. Anomalous changes in seed germination percentage (as a function of irradiation dose) were caused by the redistribution of irradiated seeds between fractions I and II. The measurements of room temperature phosphorescence in air-dry seeds and the phosphorescence of endogenous porphyrines of imbibing seeds showen that the germination decrease after the irradiation with low doses (19 cGy and 3 Gy) was caused by the increase in the number of weak seeds of fraction II, which had high rates of water uptake and suffered from hypoxia under seed coat. Some of these seeds suffocated from hypoxia, and other seeds produced seedlings with morphological defects (such seeds were considered incapable of germination). During storage of seeds irradiated at doses 19 cGy-10 Gy, the recovery of germination percentage (after its initial decrease) was caused by the decrease in seed number in fraction II. The subsequent germination decrease was caused by seed death. The higher was the irradiation dose, the faster were changes in germination percentage during storage of irradiated seeds. Bimodal changes in pea seed germination with the increase of y-irradiation dose has apparently the same origin as the changes in seed germination during accelerated ageing.     

Lipopolysaccharide containing L-acofriose in the filamentous blue-green alga Anabaena variabilis

For the first time, an O-antigenic lipopolysaccharide (LPS) has been isolated from a filamentous blue-green alga (Anabaena variabilis). It was extractable with phenol-water, resulting in extraction of the bulk of the LPS into the phenol phase. The polysaccharide moiety of this LPS consists of l-rhamnose, its 3-O-methyl ether l-acofriose, d-mannose, d-glucose, and d-galactose. l-Glycero-d-mannoheptose and 2-keto-3-deoxyoctonate, the two characteristic sugar components of enteric LPS, and phosphate groups are absent from the A. variabilis O antigen. The only amino sugar present is d-glucosamine. Three hydroxy fatty acids were identified, namely, beta-hydroxymyristic, beta-hydroxypalmitic and beta-hydroxystearic acids, in addition to palmitic and unidentified fatty acid. The LPS of A. variabilis is localized in the outermost cell wall layer and behaves like a bacterial O antigen in serological tests. The passive hemagglutination yielded high titers with isolated LPS (pretreated by heat or by alkali) and rabbit antisera prepared against living or heat-killed cells. The position of the precipitation arcs after immunoelectrophoresis of the O antigen indicates the lack of charged groups. The water phase of the phenol-water extract contains, in high yield, a glucose polymer. It is serologically inactive as shown by the passive hemagglutination test and by agar-gel precipitation.     

Effect of cytokines and lipopolysaccharide on CD14 antigen expression in human monocytes and macrophages.

The 52 kD myeloid membrane glycoprotein CD14 represents the receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein (LBP); it is involved in LPS induced tumor necrosis factor-alpha production. Expression of CD14 increases in monocytes differentiating into macrophages, and it is reduced by rIFNg in monocytes in vitro. In the present study CD14 membrane antigen expression was investigated in cultures of human mononuclear leucocytes (PBL), in elutriated, purified monocytes, and in blood monocyte derived Teflon cultured macrophages. Cells were incubated for 15 or 45 h with rIL-1, rIL-2, rIL-3, rIL-5, rIL-6, rTNFa, rGM-CSF, rM-CSF, rTGFb1, rIFNa, lipopolysaccharide (LPS), and, as a control, rIFNg. The monoclonal antibodies Leu-M3 and MEM 18 were used for labelling of CD14 antigen by indirect immunofluorescence and FACS analysis of scatter gated monocytes or macrophages. IFNg concentrations were determined in PBL culture supernatants by ELISA. rIFNa and rIL-2 reduced CD14 in 15 and 45 h PBL cultures, an effect mediated by endogenous IFNg, since it was abolished by simultaneous addition of an anti-IFNg antibody. rIFNa and rIL-2 were ineffective in purified monocytes or macrophages. rIL-4 strongly reduced CD14 in PBL and purified monocytes after 45 h, whereas in macrophages the decrease was weak, although measurable after 15 h. The other cytokines investigated did not change CD14 antigen expression. Cycloheximide alone reduced CD14, but when added in combination with rIFNg the effect on CD14 downregulation was more pronounced. The effect of rIFNg on CD14 in PBL cultures was dose-dependently inhibited by rIL-4 and this inhibition is probably due to an IL-4 mediated blockade of IFNg secretion. LPS at a low dose increased CD14, at a high dose it produced a variable decrease of CD14 in PBL, which was probably due to LPS induced IFNg secretion. LPS strongly enhanced CD14 in 45 h cultures of purified monocytes. The results, showing that CD14 antigen expression is upregulated by LPS and downregulated by rIFNg and rIL-4, suggest that the LPS-LBP receptor is involved in the feedback response of IFNg and IL-4 to LPS stimulation.     

嗜肺巴氏杆菌外膜蛋白和脂多糖抗原在血清学诊断中的意义

目的评价嗜肺巴氏杆菌外膜蛋白(OMP)和脂多糖(LPs)作为血清学诊断抗原的敏感性和特异性.方法用OMP、LPS和全菌(WC)作为Western blot和ELISA的诊断抗原检测自然感染和实验感染嗜肺巴氏杆菌小鼠相应的IgG抗体滴度,同时测定3种抗原与实验动物常见致病菌的交叉反应.结果与嗜肺巴氏杆菌自然感染和实验感染小鼠血清的ELISA反应中,不同时期,LPS作为诊断抗原时血清抗体阳性率最高,WC次之,OMP最低.自然感染小鼠群中,出生4周LPS抗体阳性率即可达80%,而同期的WC和OMP仅为25%和20%,故LPS敏感性最高.与实验动物常见致病菌免疫血清和阴性种鼠血清的ELISA反应中,WC抗原表现出较高的吸光度(A)值,经Western blot证实,其反应为非特异性反应,LPS抗原特异性最强,OMP抗原次之.结论混合多株具有型或种特异性的OMP或LPS作为ELISA的诊断抗原,无论从特异性和敏感性上均高于全菌抗原.     

Selenium Pretreatment Enhances the Radioprotective Effect and Reduces the Lethal Toxicity of Wr-2721

Although WR-2721, S-2-(3-aminopropylamino)ethylphosphorothioic acid, is an effective radioprotector, its use is limited by its toxicity. Combining WR-2721 with other agents might decrease its toxicity and/or increase its effectiveness. The effect of selenium (Se) pretreatment on the acute toxicity and radioprotective effect of WR-2721 was studied in male CD2F1 mice. Injection of 1.6mg/kg Se 24 hr before WR-2721 (800-1200 mg/kg, IP) decreased the lethality of WR-2721 significantly. Lower doses of Se were also effective, but simultaneous administration was not effective. Se injection alone (1.6 mg/kg) 24 hr before cobalt-60 irradiation increased the survival (dose reduction factor, DRF = 1.1) significantly. A synergistic effect on post-irradiation survival was observed when Se was injected 24 hr before WR-2721 (200-600 mg/ kg IP before irradiation). For example, after exposure to 22 Gy (1 Gy/min), 30-day survival was 100% when mice were treated with both Se and 600mg/kg WR-2721, and was 13% with WR-2721 alone. The DRF after 400 mg/kg WR-2721 was 2.6 with Se compared to 2.2 without Se pretreatment. Alkaline phosphatase activity in bone marrow cells and serum was significantly depressed after treatment with 1.6 mg/kg Se, suggesting that a retardation of conversion of WR-2721 to its active free sulfhydryl form through the action of alkaline phosphatase might be partly responsible for the effects of Se. Other possible mechanisms related to the antioxidant properties of Se are under investigation.