A comparison of the glutathione S-transferases of trout and rat liver.

1. Cytosol from trout liver, gills and intestinal caeca has substantial glutathione S-transferase activity. 2. Gel-exclusion and ion-exchange chromatography suggest that trout liver has several glutathione S-transferases with different molecular weights and ionic charges. 3. A component capable of binding lithocholic acid eluted together with glutathione S-transferase activity. Some of the transferase activity did not elute together with binding activity. 4. The enzymic activity from trout liver was less stable at 37 degrees C than that from rat liver. 5. The glutathione S-transferases of fish liver have a similar specific activity to those of rat liver but different molecular properties.     

Toxoplasma gondii in wild and domestic animals from New Caledonia

Samples (serum or meat juice) collected from 205 animals in New Caledonia in April 2009 were tested for antibodies against Toxoplasma gondii by ELISA using the multi-species ID Screen^® Toxoplasmosis Indirect kit (IDVET, Montpellier). Antibodies to T. gondii were detected in 2% (1/49) of the pigs, in 3.3% (1/30) of the cattle, in 13.8% (4/29) of Rusa deers, in 16% (4/25) of the horses, in 32.8% (21/64) of the dogs, and in 50% (4/8) of cats. Statistically, no significant difference was observed between T. gondii seroprevalence and age or sex. No survey on the prevalence of T. gondii in animals has ever been conducted in New Caledonia and this is the first serological evidence of T. gondii in Rusa deer (Cervus timorensis russa). These results indicate an important circulation of T. gondii exists in the animal populations of New Caledonia. In view of humans being exposed, it is advisable to insist on sanitary education and on respect for good hygienic and food practice.     

Acidic and basic forms of glutathione S-transferases from human placenta and comparison with human kidney glutathione S-transferase

Glutathione S-transferase (GSH-transferase) was purified from human placenta and kidney by affinity chromatography on S-glutathione-carbamidomethyl-epsilon-aminolysyl-Sepharose CL 4B and gel filtration chromatography on Sephades G-75. Electrophoretically pure enzyme with the specific activities of 50.7 and 55.9 U/mg, respectively, were obtained. In addition to the known acidic isoenzyme from human placenta (isoelectric point, pI, 4.5), we describe here for the first time the presence of 6 basic forms with pI values between 8.0 and 9.0. The kidney GSH-transferase contained 2 acidic forms with isoelectric points at 4.6 and 4.65, and 6 basic forms with pI values between 8.7 and 9.4. The basic and acidic isoenzymes from placenta were separated by ion exchange chromatography on Sephadex DEAE A-25. The acidic form accounted for 36% of the total GSH-transferase activity from placenta. Antibodies against the kidney enzyme were raised in rabbit. Total cross-reactivity of placental GSH-transferase with antikidney-GSH-transferase antibodies was obtained, suggesting that the kidney and placental enzymes are immunologically closely related.     

Seroprevalence of Anaplasma phagocytophilum in domestic and wild animals from central Italy

From January 2004 to July 2007, 2455 sera were collected from domestic and wild animals living in central Italy and tested by indirect immunofluorescence assay to detect antibodies against Anaplasma phagocytophilum. Considering sera with 1:40 antibody titers as positive, 336 (13.68%) animals scored positive. The percentages of seropositivity were: 46.26% (31/67) in fallow deer, 46.15% (24/52) in red deer, 16.89% (134/793) in horses, 16.78% (23/137) in cattle, 12.74% (13/102) in sheep, 8.76% (108/1232) in dogs, 4.16% (3/72) in goats. These data confirm the presence of A. phagocytophilum in wild ruminants and domestic animals, including pets, in central Italy.     

Transmission of Neospora caninum between wild and domestic animals

To determine whether deer can transmit Neospora caninum, brains of naturally infected white-tailed deer (Odocoileus virginianus) were fed to 4 dogs; 2 of these dogs shed oocysts. Oocysts from 1 of the dogs were tested by polymerase chain reaction and found to be positive for N. caninum and negative for Hammondia heydorni. The internal transcribed spacer 1 sequence of the new strain (designated NC-deer1) was identical to N. caninum from domestic animals, indicating that N. caninum is transmitted between wild and domestic animals, often enough to prevent divergent evolution of isolated populations of the parasite. NC-deerl oocysts were administered to a calf that developed a high antibody titer, providing evidence that N. caninum from wildlife can infect cattle. In addition, N. caninum antibody seroprevalence was detected in 64/164 (39%) free-ranging gray wolves (Canis lupus), 12/113 (11%) coyotes (Canis latrans), 50/193 (26%) white-tailed deer, and 8/61 (13%) moose (Alces alces). These data are consistent with a sylvatic transmission cycle of N. caninum between cervids and canids. We speculate that hunting by humans favors the transmission of N. caninum from deer to canids, because deer carcasses are usually eviscerated in the field. Infection of canids in turn increases the risk of transmitting the parasite to domestic livestock.     

Purification and characterization of glutathione S-transferases from rat pancreas

Glutathione S-transferases (GSTs) of rat pancreas have been characterized and their interrelationship with fatty acid ethyl ester synthase (FAEES) has been studied. Seven GST isozymes with pI values of 9.2, 8.15, 7.8, 7.0, 6.3, 5.9 and 5.4 have been isolated and designated as rat pancreas GST suffixed by their pI values. Structural, immunological and kinetic properties of these isozymes indicated that GST 9.2 belonged to the alpha class, GST 7.8, 7.0, 6.3 and 5.9 belonged to the mu class, whereas GST 8.15 and 5.4 belong to pi class. The N-terminal sequences and pI values of the mu class isozymes suggested that rat GST subunits 3, 4 and 6 may be expressed in pancreas. N-Terminal sequences of both the pi class isozymes, GST 8.15 and 5.4, were similar to that of GST-P, but there were significant differences in the substrate specificities of these two enzymes. Results of peptide finger print studies also indicated minor structural differences between these two isozymes. None of the GST isozymes of rat pancreas expressed FAEES activity. Rat pancreas had a significant amount of FAEES activity, but it segregated independently during the purification of GST indicating that these two activities are expressed by different proteins and are not related as suggested previously.     

Characterization of glutathione S-transferases from day-old chick livers

Glutathione S-transferases (GSTs, EC 2.5.1.18) were isolated from the liver cytosolic fraction of 1 day old Leghorn chicks by S-hexylglutathione and glutathione affinity columns arranged in tandem. After sample loading, the affinity columns were detached from each other and developed separately. Four groups of GSTs (CL 1, 2, 3, and 4) were eluted from the hexylglutathione column, and an additional group of GSTs (CL 2 and 5) was eluted from the glutathione affinity column. CL 2, CL 3, and CL 5 were further purified to homogeneity by chromatofocusing, and the substrate specificities of each group were determined. Fractions from the chromatofocusing column were analyzed by native IEF electrophoresis. Protein bands were electroblotted onto PVDF membrane for N-terminal sequence analysis or extracted from IEF gel and rerun on SDS-PAGE to determine the subunit composition of each GST dimer. CL 2, CL 3, and CL 5 can form homodimers, whereas CL 1 and CL 4 exist only as CL 1-2 and CL 3-4 heterodimers. CL 2 and CL 5 have N-terminal amino acid sequences homologous to rat liver Yb and Ya GSTs, respectively. CL 1 has a unique N-terminal sequence that is not homologous to any known GSTs.     

Identification of multiple glutathione S-transferases from Daphnia magna

1. Six anionic glutathione S-transferases (GST) were purified from the crustacean, Daphnia magna, by means of affinity chromatography, that are responsible for ca. 40% of cytosolic GST activity. 2. Electrophoresis in the presence of sodium dodecyl sulfate (SDS) revealed the presence of three proteins, with molecular weights of 27,500, 28,000, and 30,200. 3. Separation under nondenaturing conditions revealed six proteins, all of which exhibited GST activity, with molecular weights ranging from 55,000 to 61,700. 4. Ethacrynic acid is a competitive inhibitor of activity towards CDNB of all six GSTs, binding each with similar affinities. 5. Chlorinated phenols are also competitive inhibitors of the enzyme, with the degree of inhibition being directly correlated with the lipophilicity of the compounds. 6. Analysis of inhibition of separated isoforms reveals that form 4 is most strongly inhibited by these chlorinated phenols, with forms 5 and 6 being inhibited to a lesser degree.     

Trialkyl phosphorothioates and glutathione S-transferases

Using a rat liver cytosol source of enzyme trialkyl phosphorothioates have been shown to be substrates of glutathione S-transferases. Using OSS-trimethyl phosphorodithioate (OSS-Me(O] and OOS-trimethyl phosphorothioate (OOS-Me(O] the methyl transferred to the sulphydryl of glutathione is that attached to phosphorus via an oxygen atom. Fractionation of liver cytosol has shown that although the bulk activity is due to the three isozymes (1-1; 3-4; 1.2), OSS-Me(O) is a general substrate for glutathione S-transferases. The specific activity is low compared with the substrates 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene.     

Purification and characterization of glutathione S-transferases from guinea pig liver

Four types of glutathione S-transferase were purified to homogeneity from guinea pig liver by DEAE-cellulose, Sephadex G-75, CM-cellulose, and affinity chromatography. These isozymes were named a, b, c, and d based on the reverse order of elution from a CM-cellulose column, and had specific activities of 89.6, 92.2, 99.0, and 44.0 units/mg, respectively, when assayed with 1 mM each of 1-chloro-2,4-dinitrobenzene and reduced glutathione. All four transferases of guinea pig liver were homodimers. The transferases b, c, and d had a similar molecular weight of 50,000 and their subunit sizes were 25,000, but the corresponding values for transferase a were 45,000 and 23,500, respectively. Transferase a was notably different in the activities towards organic hydroperoxides and 1,2-dichloro-4-nitrobenzene from the other isozymes. Transferases a and b, the major forms in guinea pig liver, were studied with respect to their biochemical properties, including kinetic parameters, absorption and fluorescence spectra, and bilirubin binding. Glutathione peroxidase activity of the transferase a was about 100 times higher than that of other isozymes. In guinea pig liver, it is estimated that transferase a is the major glutathione peroxidase, accounting for about 75% of the total organic hydroperoxide reduction.     

Indomethacin inhibition of glutathione S-transferases

Indomethacin inhibited rat liver glutathione S-transferases (EC 2.5.1.18). Its inhibition was non-competitive with respect to 3,4-dichloronitrobenzene with an apparent Ki of 5.3 X 10(-5) M and uncompetitive with respect to glutathione with an apparent Ki of 4.0 X 10(-5) M. 4-Chlorobenzoic acid and 5-methoxy-2-methylindole-3-acetic acid, two metabolites of indomethacin, were weak inhibitors of the enzymes. On the other hand, meclofenamic acid was a competitive inhibitor of the enzymes with an apparent Ki of 3.0 X 10(-4) M. Possible significance of these findings in arachidonic acid metabolism is discussed.     

Isoelectric focusing of glutathione S-transferases from rat liver and kidney

The glutathione S-transferases that were purified to homogeneity from liver cytosol have overlapping but distinct substrate specificities and different isoelectric points. This report explores the possibility of using preparative electrofocusing to compare the composition of the transferases in liver and kidney cytosol. Hepatic cytosol from adult male Sprague–Dawley rats was resolved by isoelectric focusing on Sephadex columns into five peaks of transferase activity, each with characteristic substrate specificity. The first four peaks of transferase activity (in order of decreasing basicity) are identified as transferases AA, B, A and C respectively, on the basis of substrate specificity, but the fifth peak (pI6.6) does not correspond to a previously described transferase. Isoelectric focusing of renal cytosol resolves only three major peaks of transferase activity, each with narrow substrate specificity. In the kidney, peak 1 (pI9.0) has most of the activity toward 1-chloro-2,4-dinitrobenzene, peak 2 (pI8.5) toward p-nitrobenzyl chloride, and peak 3 (pI7.0) toward trans-4-phenylbut-3-en-2-one. Renal transferase peak 1 (pI9.0) appears to correspond to transferase B on the basis of pI, substrate specificity and antigenicity. Kidney transferase peaks 2 (pI8.5) and 3 (pI7.0) do not correspond to previously described glutathione S-transferases, although kidney transferase peak 3 is similar to the transferase peak 5 from focused hepatic cytosol. Transferases A and C were not found in kidney cytosol, and transferase AA was detected in only one out of six replicates. Thus it is important to recognize the contribution of individual transferases to total transferase activity in that each transferase may be regulated independently.     

Serotyping of Campylobacter jejuni, Campylobacter coli, and Campylobacter laridis from domestic and wild animals

By using 50 unabsorbed antisera, we were able to serotype 272 (65.7%) of 414 thermotolerant campylobacters from wild and domestic animals, on the basis of heat-stable antigens identified by means of passive hemagglutination. Forty-two serotypes were recognized. The pattern of serotypes detected in the various animal species was compared to human clinical isolates by using the Czekanowski index (proportional similarity index). The highest degree of similarity to the clinical isolates was observed for the poultry isolates, followed by strains from wild birds, flies, and pigs (in order of decreasing similarity). The serotypes recovered most frequently from poultry (LAU 1 and LAU 2) were also most prevalent in Norwegian patients. In contrast, serotype LAU 35/44, the predominant porcine serotype, was never recovered from human clinical specimens. Flies captured in chicken farms and in piggeries harbored serotypes which were also commonly seen in chickens and pigs, respectively. Nine of the strains included in this study could not be ascribed to any defined species. All of these were resistant to nalidixic acid and did not produce H2S.     

Purification and characterization of the individual glutathione S-transferases from sheep liver

The glutathione S-transferases (EC 2.5.1.18) have been purified to electrophoretic homogeneity from 105,000g supernatant of sheep liver homogenate by employing a combination of gel filtration on Sephadex G-150 and affinity chromatography on S-hexylglutathione-linked Sepharose-6B columns. Approximately 70% of the original glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity toward cumene hydroperoxide could be recovered by this purification method. Of particular importance in developing this procedure was the fact that the enzyme preparation obtained after affinity column chromatography represented all the isozymes of sheep liver glutathione S-transferases. Further purification by CM-cellulose and DEAE-cellulose column chromatography resolved the glutathione S-transferases into seven distinct cationic isozymes designated C-1, C-2, C-3, C-4, C-5, C-6, and C-7 and five overlapping anionic transferases designated A-1, A-2, A-3, A-4, and A-5, respectively, in the order of their elution from the ion-exchange columns. The sodium dodecyl sulfate SDS-gel electrophoretic data on subunit composition revealed that cationic enzymes are composed of two subunits with an identical Mr of 24,000 whereas a predominant subunit with Mr of 26,000 was observed in all anionic isozyme peaks except A-1. Cationic isozymes accounted for approximately 98% of the total peroxidase activity associated with the glutathione S-transferase whereas only A-1 of the anionic isozymes displayed some peroxidase activity. Isozyme C-4 was found to be the most abundant glutathione S-transferase in the sheep liver. Characterization of the individual transferases by their specificity toward a number of selected substrates, subunit composition, and isoelectric points showed some similarities to those patterns for human liver glutathione S-transferases.     

Resolution and kinetic characterization of glutathione S-transferases from human jejunal mucosa

Cytosolic glutathione S-transferases were purified from human jejunal mucosa by affinity chromatography on S-hexylglutathione-Sepharose 4B. Chromatofocusing in the pH range 7-4 yielded peaks with apparent pI's of 7.2 (peak 1), 5.2 (peak 2), and 4.4 (peak 3). Each enzymatic fraction was shown to have a homodimeric structure, with subunit mass of 24.9 +/- 0.5 (P1), 27.9 +/- 0.9 (P2), and 23.4 +/- 0.8 (P3) kDa, as determined by SDS-PAGE. The substrate specificity of each peak was tested using discriminating substrates for basic, near-neutral, and acidic GSTs. With cumene hydroperoxide, the diagnostic substrate for the alpha (basic) class of GSTs, P1 showed 8- to 36-fold higher activity than P2 and P3. Ethacrynic acid, the selective substrate for the acidic enzyme (pi), gave highest activity with P3. The inhibitory potentials of sulfobromophthalein, cibacron blue, tributyltin acetate, triphenyltin chloride, and bromphenol blue were also tested. A qualitative resemblance between P1 and alpha, and P3 and pi GSTs was noted. The substrate specificity and inhibiton parameters of P2 corresponded most closely to those of mu-GST. The relative abundances of P1, P2, and P3 (based on CDNB-conjugating activity) were 35, 5, and 60%, respectively.