Tissue distribution of monomeric glutathione peroxidase in broiler chicks.

It is known that there are two kinds of enzymes which show glutathione peroxidase activity, 'classical' glutathione peroxidase and glutathione S-transferase. Recently, a third enzyme was found, monomeric glutathione peroxidase, in broiler chick liver cytosolic fraction. To gain an insight into the possible physiological role of the monomeric glutathione peroxidase, the distribution of this enzyme in other broiler tissues was studied. The monomeric glutathione peroxidase was found in all tissues examined and in erythrocytes. The percentage of the total glutathione peroxidase activity accounted for by the monomeric enzyme ranged from 4 per cent in erythrocytes to 28 per cent in liver. Livers from three avian and two mammalian species contained the monomeric glutathione peroxidase. The contribution of the monomeric enzyme to total glutathione peroxidase activity with cumene hydroperoxide was high in poultry livers, while only trace monomeric glutathione peroxidase activity was found in mammalian livers. Chick monomeric glutathione peroxidase showed high activity toward phospholipid hydroperoxide. Thus, monomeric glutathione peroxidase might be an important enzyme in reducing membrane lipid hydroperoxides in birds.     

Effect of chemicals on glutathione peroxidase of chick liver

Chick liver glutathione peroxidase activity was separated into selenium-dependent and selenium-independent enzyme fractions and the effects of chemicals on the activities of each fraction were examined. Clofibrate induced the selenium-dependent enzyme, while phenobarbital, butylhydroxyanisole and trans-stilbene oxide elevated selenium-independent peroxidase activity. A third enzyme, which showed glutathione peroxidase activity toward both hydrogen peroxide and cumene hydroperoxide, was found.     

Oxidative stress and bovine liver diseases: role of glutathione peroxidase and glucose 6-phosphate dehydrogenase

This article summarizes the different types of free radicals, antioxidants and the effect of oxidative stress on the activities of glutathione peroxidase and glucose 6-phosphate dehydrogenase in bovine liver diseases. A growing body of evidence suggests that the formation of reactive oxygen species is a common occurrence associated with most if not all disease processes. The overall importance of reactive oxygen species to the progression and severity of various disease states varies greatly depending on the conditions and whether the disease is acute or chronic. Free radical researches in animals are in progress and further investigations are needed to establish the involvement of reactive oxygen species in diseases affecting different animal species and the pathology they produce.     

Prostaglandins and glutathione peroxidase in bovine mastitis

Prostaglandin (PG) levels in milk and plasma samples from mastitic cows were determined by radioimmunoassay and compared with erythrocyte glutathione peroxidase (GSH-Px) and other relevant parameters in milk and blood. The overall levels of PGE2, PGF2 alpha and thromboxane B2 (TXB2) in milk were two to four times higher than in blood plasma both in healthy and diseased animals (P less than 0.01). In mastitic milk the PG levels were 24 to 55 per cent and in blood plasma 41 to 95 per cent higher than in healthy animals. The changes were significant and largest for the PGF2 alpha values. In milk, the PG concentrations correlated with the markedly elevated cell count (r = 0.49 to 0.57), and TXB2 values also correlated with milk yield. In blood, PGE2 and PGF2 alpha correlated positively with serum albumin, and PGE2 also correlated with glutathione (GSH). PGE2 and PGF2 alpha correlated negatively with GSH-Px and gamma-glutamyl transferase. The substantial decline in GSH-Px in mastitic animals (P less than 0.01) may be related to changes in lipid peroxidation and PG formation. The possible implications of these findings in the treatment of mastitis are discussed.     

Purification and initial characterisation of koala immunoglobulins

The production of koala immunoglobulins (Ig) was elicited by the immunisation of a koala (Phascolarctos cinereus) with bovine serum albumin. This Ig was then purified using the highly specific techniques of affinity chromatography. The purified protein was compared with a "potential" koala Ig protein which was subsequently purified by Protein G chromatography and both proteins were further characterised by agarose/SDS-PAGE and immunoelectrophoresis. Results indicate that koalas do produce Ig in response to antigen challenge, koala Ig has a higher net negative charge than that seen in most other mammals and possibly two subclasses of IgG and IgM are present in normal koala serum.     

Expression and characterisation of a Psoroptes ovis glutathione S-transferase

The astigmatid mite Psoroptes ovis is the causative agent of sheep scab, a highly contagious parasitic disease of sheep. Infection causes severe allergic dermatitis, resulting in damage to the fleece and hide, loss of condition and occasional mortality. Interest in the P. ovis allergens led us to characterise a glutathione S-transferase (GST) which displays homology to GST allergens isolated from the house dust mite, Dermatophagoides pteronyssinus and the cockroach, Blatella germanica. A cDNA encoding a mu-class GST from P. ovis was expressed in Escherichia coli and the recombinant protein purified for biochemical analysis. SDS-PAGE analysis indicated that the purified product was homogeneous and had an apparent molecular weight of 30 kDa. The recombinant GST (rGST) is active towards the substrate 1-chloro-2,4-dinitrobenzene (CDNB), whereas 1,2-dichloro-4-nitrobenzene (DCNB) is a poor substrate. The recombinant protein was also tested for recognition by IgE and IgG antibodies in serum from P. ovis na?ve and P. ovis infested sheep. Neither IgE nor IgG antibodies were detected to the rGST. Prausnitz--Küstner testing with rGST did not provoke a characteristic weal and flare response. Biopsies collected at the PK test sites were stained for eosinophils, neutrophils, mast cells and basophils. Neutrophil, mast cell and basophil counts were not significantly different to the controls. Eosinophil numbers were significantly higher than controls, but were not due to an IgE response.     

Serum glutathione peroxidase activity and blood selenium in pigs

Blood serum glutathione peroxidase activity and blood selenium concentration were measured in blood samples from pigs subjected to experimentally induced selenium deficiency and dietary selenium supplementation on graded levels. A highly significant correlation between blood selenium and serum GSH-Px activity in pigs, especially in selenium deficient pigs, was demonstrated. There was also a strong relationship between blood selenium concentration and serum GSH-Px activity in pigs receiving dietary selenium at graded levels. Serum GSH-Px activity exhibited an excellent close-response relationship to dietary selenium. Linear regression analysis showed that the increased serum GSH-Px activity was a function of the dietary selenium concentration. The fitness of serum in monitoring slight changes of the selenium status of pigs with help of the estimation of GSH-Px activity was discussed. The measurement of serum GSH-Px activity seems to provide a useful and rapid means for defining selenium requirements and for identifying selenium deficiency in growing pigs.     

Piglet blood glutathione peroxidase levels and preweaning mortality.

The blood glutathione peroxidase levels of one day old piglets from 22 litters were examined. Body weight and piglet survival were monitored in order to assess the relationship between these two factors and blood glutathione peroxidase activity. The mean blood glutathione peroxidase level of one day old piglets (65 mu/gHb) was significantly lower (p0.001) than the mean level (85 mu/gHb) at weaning. The mean blood glutathione peroxidase activity of one day old piglets was not related to the size of the litter, but was related (p less than 0.1) to the mean litter blood glutathione peroxidase level at weaning time. Piglet blood glutathione peroxidase was not related to piglet body weight. The blood glutathione peroxidase level of the sows at one-day post-farrowing was not related to the mean blood glutathione peroxidase activity of their litters at one day of age but was correlated (p less than 0.1) with the mean blood glutathione peroxidase levels of their litters at weaning. Piglet viability was shown to be strongly correlated (p less than 0.001) with body weight at one day of age. The blood glutathione peroxidase level of one day old piglets was weakly associated (p less than 0.1) with piglet survival. Further work is required to clarify this latter observation, which suggests that selenium supplementation to newborn piglets may be beneficial regardless of the dams nutritional status.     

Biological characterisation of Australian isolates of chicken anaemia agent

Three Australian isolates of chicken anaemia agent (CAA) resisted treatment at 70 degrees C for 5 min and chloroform treatment. Although minor antigenic differences were detected using monoclonal antibodies to CAA, the Australian isolates were indistinguishable from the reference Cux-1 and Gifu-1 isolates in cross-immunofluorescence and cross-neutralisation tests employing polyclonal chicken antiserums. The Australian viruses were pathogenic for intramuscularly inoculated 1-day-old SPF chicks, but were less pathogenic for 7-day-old chicks. Thus the Australian isolates of CAA did not differ significantly in these properties from previously characterised CAA isolates from other continents.     

Total glutathione and glutathione reductase in bovine erythrocytes and liver biopsy

The goal of the present study was to measure the total glutathione level and glutathione reductase activity in bovine erythrocytes and liver biopsy. Five cows were injected intraperitoneally with DL-ethionine (12.5 mg/kg B.W.), and two control cows were injected with normal saline (0.9% NaCl). Ultrasonography guided liver biopsy, and blood samples were collected at 0, 4, 7 and 10 days after injection. The hepatic total glutathione level was significantly increased on Days 7 (p<0.05) and 10 (p<0.01), and hepatic glutathione reductase activity was significantly increased on Days 4 (p<0.05), 7 (p<0.01) and 10 (p<0.01). There were insignificant changes in the erythrocytic total glutathione level. The present study demonstrated that liver biopsy is a valuable tool for detecting oxidative stress and for diagnosing hepatic dysfunction in cattle from the viewpoint of the status of glutathione and glutathione reductase.     

Assessment of blood glutathione peroxidase activity in the dromedary camel

Blood glutathione peroxidase (GSH-Px) levels in 709 normal dromedary camels (442 females and 267 males) were assessed in the Canary Islands. All animals were intensively reared, and three different nutritional systems were evaluated, depending on selenium content of the diet. Mean GSH-Px level in the total population was 288.5+/-157.2 IU x g(-1) Hb. Reference ranges were estimated and enzymatic activities below 51 IU x g(-1) Hb were considered inadequate. GSH-Px activities obtained in females (298.1+/-155.7 IU x g(-1) Hb) were significantly (P = 0.037) higher than in males (272.6+/-157.2 IU x g(-1) Hb). When age groups were compared, only males between 6 and 12 months old exhibited significantly lower mean GSH-Px (P = 0.006) than females. A high correlation (r = 0.88) between serum selenium concentration and blood GSH-Px activity was estimated, and the regression equation was y = 2.5101x + 42.423. Selenium content of the diet above 0.1 mg x kg(-1) DM seems to supply adequate selenium requirements for dromedaries under intensive husbandry.     

Production and characterisation of monoclonal antibodies specific for chicken interleukin-12

Using genetic immunisation of mice, we produced antibodies against chicken interleukin-12p40 (chIL-12p40), also known as IL-12β. After a final injection with a recombinant chIL-12p40 protein, several stable hybridoma cell lines were established which secreted monoclonal antibodies (mAbs) to this component of the heterodimeric IL-12 cytokine. Specific binding of three of the mAbs to COS-7 cell-derived recombinant chIL-12p40 and the chIL-12p70 heterodimer was demonstrated in an indirect ELISA, and in dot blots. Two of the mAbs were used to develop a capture ELISA, suitable for detecting both recombinant protein (chIL-12p40 and the heterodimeric p70 protein) and native chIL-12. The mAbs were further characterised to show utility in immunocytochemistry.     

Serum-free culture of adult chicken hepatocytes; morphological and biochemical characterisation

The morphological and biochemical characterisation of adult chicken hepatocytes in a serum-free culture are described. When cultured in positively charged plastic dishes, chicken hepatocytes formed a monolayer cell sheet. The monolayer morphology of these chicken hepatocytes was quite distinct from the spheroid shape of rat hepatocytes cultured under similar conditions. Electron microscopy showed that the cytoplasmic organelles of chicken hepatocytes were well preserved in vitro. Two-dimensional gel electrophoresis showed that the chicken hepatocytes secreted liver-specific proteins. Several enzymes of glucose-6-phosphatase, cytochrome P-450 or glutathione S-transferase, involved in metabolic and biotransformation pathways in the liver, were retained in the chicken hepatocytes in a serum free condition. These findings suggest that the primary culture of adult chicken hepatocytes with a serum-free culture system could be useful to study the hepatic metabolic pathway in the chicken and its response to various chemicals.     

Characterisation of an antiserum and development of an ELISA for glutathione peroxidase

Sheep red blood cells were fractionated by ion exchange and gel filtration chromatography to yield glutathione peroxidase approximately 99% pure. An antiserum against glutathione peroxidase was raised in the rabbit. The antiserum has been shown to cross-react with both bovine and human glutathione peroxidase by double diffusion.An enzyme linked immunosorbent assay has been developed for glutathione peroxidase which detected 6.15×10^–5 IU of the enzyme. The antiserum has also been shown to be effective in the detection of glutathione peroxidase immobolised on strips of nitrocellulose, subsequent to sodium dodecyl sulphate polyacrylamide gel electrophoresis, by second antibody conjugate. Avidin-biotin was also used to detect nitrocellulose immobolised enzyme. These techniques provide an alternative highly sensitive and specific means of assaying glutathione peroxidase which is not dependent on the lability of enzymatic activity nor the chemical specificity of the assay.     

Characterisation of an antiserum and development of an ELISA for glutathione peroxidase

Sheep red blood cells were fractionated by ion exchange and gel filtration chromatography to yield glutathione peroxidase approximately 99% pure. An antiserum against glutathione peroxidase was raised in the rabbit. The antiserum has been shown to cross-react with both bovine and human glutathione peroxidase by double diffusion. An enzyme linked immunosorbent assay has been developed for glutathione peroxidase which detected 6.15 X 10(-5) IU of the enzyme. The antiserum has also been shown to be effective in the detection of glutathione peroxidase immobolised on strips of nitrocellulose, subsequent to sodium dodecyl sulphate polyacrylamide gel electrophoresis, by second antibody conjugate. Avidin-biotin was also used to detect nitrocellulose immobolised enzyme. These techniques provide an alternative highly sensitive and specific means of assaying glutathione peroxidase which is not dependent on the lability of enzymatic activity nor the chemical specificity of the assay.     

The effect of tilmicosin on cardiac superoxide dismutase and glutathione peroxidase activities

In this study, the effect of tilmicosin on cardiac superoxide dismutase and glutathione peroxidase activities was investigated. Forty male BALB/c mice were used as material. Ten mice served as a control group, and 30 mice were injected with tilmicosin (25 mg/kg body weight, subcutaneously, with a single injection). After drug administration, they were monitored for 3 days. Tilmicosin caused decreases in cardiac superoxide dismutase and glutathione peroxidase activities.     

Production and characterisation of monoclonal antibodies specific for chicken interleukin-2.

Using genetic immunisation of mice, we produced antibodies against chicken interleukin-2 (ChIL-2), the first produced against a non-mammalian interleukin. After a final injection with a recombinant ChIL-2 protein, two stable hybridoma cell lines were established which secreted monoclonal antibodies (MAbs) against this cytokine. Specific binding of the two MAbs to recombinant ChIL-2 produced by Escherichia coli and COS-7 cells was demonstrated in an indirect ELISA, Western blotting and dot blots. Both of them were able to neutralise the biological activity of the ChIL-2, but neither allowed the detection of ChIL-2 by flow cytometry.