Purification and characterisation of chicken liver monomeric glutathione peroxidase

1. A novel glutathione peroxidase, which is distinct from tetrameric glutathione peroxidase, was purified to homogeneity from a broiler chick liver cytosolic fraction using 5 different column chromatographic methods.

2. The enzyme in cytosol was separated from ‘classic’ tetrameric glutathione peroxidase and glutathione S‐transferases by DEAE‐Sephacel and Sephadex G‐100 chromatographies and further purified by Mono Q, hydroxylapatite and sulphobro‐mophthalein‐S‐glutathione‐agarose chromatographies.

3. The molecular weight of the purified enzyme determined by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis was 19,500 and that found by gel filtration chromatography was comparable. This indicates that the enzyme protein is a single polypeptide. The isoelectric point of the enzyme was determined as 7.0 by polyacrylamide gel isoelectric focusing.

4. The purified enzyme catalysed the reduction of hydrogen peroxide, cumene hydroperoxide, tert‐butyl hydroperoxide and linoleic acid hydroperoxide. Furthermore, it reduced phosphatidylcholine hydroperoxide in the absence of phospholi‐pase A2. The optimum pH for the enzyme reaction was 7.0. The antiserum against the purified enzyme reacted with the 19.5 kDa polypeptide in the liver cytosol of duck and quail.     

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.     

Oral administration of L-citrulline changes the concentrations of plasma hormones and biochemical profile in heat-exposed broilers

We examined the effects of oral administration of L-citrulline (L-Cit) on plasma metabolic hormones and biochemical profile in broilers. Food intake, water intake, and body temperature were also analyzed. After dual oral administration (20 mmol/head/administration) of L-Cit, broilers were exposed to a high ambient temperature (HT; 30 ± 1°C) chamber for 120 min. Oral administration of L-Cit reduced (p < .001) rectal temperature in broilers. Food intake was increased (p < .05) by heat stress, but it was reduced (p < .05) by L-Cit. Plasma levels of 3,5,3′-triiodothyronine, which initially increased (p < .0001) due to heat stress, were reduced (p < .01) by oral administration of L-Cit. Plasma insulin levels were increased by heat exposure (p < .01) and oral L-Cit (p < .05). Heat stress caused a decline (p < .05) in plasma thyroxine. Plasma lactic acid (p < .05) and non-esterified fatty acids (p < .01) were increased in L-Cit-treated heat-exposed broilers. In conclusion, our results suggest that oral L-Cit can modulate plasma concentrations of major metabolic hormones and reduces food intake in broilers.     

Isolation and purification of a protective protein antigen of Erysipelothrix rhusiopathiae

The rapid growth and high survival rate of Erysipelothrix rhusiopathiae was determined using a culture of the bacterium in tryptic soy broth supplemented with 0.3% Tris-hydroxymethyl aminomethane and 0.1% Tween 80 (TT-TS broth). High concentrations of 64, 66 and 43 kDa proteins, which are associated with protection against E. rhusiopathiae infection in mice, were obtained by alkaline treatment of whole cells using 0.05-1 N NaOH. The supernatant of alkaline treated cells (alkaline extract; AE) was stable at alkaline or neutral pH. However, aggregates appeared at neutral pH in the absence of sodium dodecyl sulphate (SDS). A high yield of 64, 66 and 43 kDa proteins was obtained from strain Agata (serovar 5). The proteins were eluted from gel bands following SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the AE from strain Agata and designated P64 and P43. The amounts of P64 and P43 isolated were 0.7 and 0.3 mg/16 g of wet bacteria, respectively. In a mouse protection test, 50% protective doses (PD50) of P64 and P43 were 0.58 and 0.63 microgram, respectively. Upon Western blotting of the AE, both anti-P64 and anti-P43 antibodies reacted with the 64 and 43 kDa proteins. From these results, it is suggested that P64 is the most effective protective antigen and that P43 (43 kDa protein) is a degradation product of P64. Therefore, the 64 kDa structural proteins are associated with the induction of a protective activity against E. rhusiopathiae infection in mice.     

Possible active transport mechanism in pharmacokinetics of flunixin-meglumin in rabbits

The plasma and urine kinetics of flunixin-meglumin (FNX, 2 mg/kg, i.v.) in rabbits were examined. Unusual pharmacokinetic profiles were obtained, including high binding percentage with plasma protein (> 99%), a short elimination half-life (< 4 hr) and a relatively large Vd-area (0.5 L/kg). These profiles indicate that some active transport mechanisms are involved in FNX disposition. The recovery of FNX from urine was approximately 9 % of the dose within 24 hr following the injection. The estimated renal clearance of the unbound drug nearly corresponded to the renal blood flow rates, indicating that active tubular secretion in the renal re-absorptive tract may be involved in the disposition. The effect of a concomitant administration of pravastatin (PV) on FNX disposition was also examined. PV is a representative substrate of a transporter in human liver cells (OATP-2). After the PV administrations, the Vd-area of FNX and total body clearance markedly decreased, indicating that FNX is actively taken up and metabolized in liver cells by an OATP-2 like transporter. In conclusion, there are at least 2 active transport pathways for FNX pharmacokinetics in rabbits, one is renal tubular secretion and the other is in the sinusoidal section of the liver.     

Induction of apoptotic lesions in liver and lymphoid tissues and modulation of cytokine mRNA expression by acute exposure to deoxynivalenol in piglets

Six 1-month-old piglets were intravenously injected with deoxynivalenol (DON) at the concentration of 1 mg/kg body weight, with three pigs each necropsied at 6 and 24 h post-injection (PI) for investigation of hepatotoxicity and immunotoxicity with special attention to apoptotic changes and cytokine mRNA expression. Histopathological examination of the DON-injected pigs revealed systemic apoptosis of lymphocytes in lymphoid tissues and hepatocytes. Apoptosis of lymphocytes and hepatocytes was confirmed by the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method and immunohistochemical staining against single-stranded DNA and cleaved caspase-3. The number of TUNEL-positive cells in the thymus and Peyer''s patches of the ileum was increased at 24 h PI compared to 6 h PI, but the peak was at 6 h PI in the liver. The mRNA expression of interleukin (IL)-1β, IL-6, IL-18, and tumor necrosis factor (TNF)-α in the spleen, thymus and mesenteric lymph nodes were determined by semi-quantitative RT-PCR, and elevated expression of IL-1β mRNA at 6 h PI and a decrease of IL-18 mRNA at 24 h PI were observed in the spleen. IL-1β and IL-6 mRNA expressions increased significantly at 6 h PI in the thymus, but TNF-α decreased at 6 h PI in the mesenteric lymph nodes. These results show the apoptosis of hepatocytes suggesting the hepatotoxic potential of DON, in addition to an immunotoxic effect on the modulation of proinflammatory cytokine genes in lymphoid organs with extensive apoptosis of lymphocytes induced by acute exposure to DON in pigs.     

RFLP analysis of a PCR amplified region of chloroplast DNA in eggplant and related Solanum species

RFLP analysis of a PCR amplified 3.2-kbp region of cpDNA bounded by the conserved sequences in rbc L and ORF 106 was performed in eggplant (Solanum melongena), its related Solanum species, S. incanum, S. virginianum (= S. surattense), S. torvum, S. aethiopicum (= S. gilo), S. aethiopicum (= S. integrifolium), S. violaceum (= S. indicum), S. violaceum (= S. sanitwongsei) and S. mammosum and the reciprocal hybrids between S. aethiopicum (= S. integrifolium) and S. melongena 'Uttara'. The target region of cpDNA was amplified correctly by PCR. The amplified products were digested with each of 10 restriction enzymes (Alu I, Ase I, BamH I, Hinf I, Msp I, Rsa I, ScrF I, Sty I, Taq I and Xba I). Variations of restriction patterns among the species were recognized after digesting the amplified products with each of the seven restriction enzymes, Taq I, Alu I, Rsa I, Sty I, Ase I, Hinf I and Xba I. The restriction patterns divided the examined nine species into the following five clusters, 1) S. melongena and S. incanum, 2) S. virginianum (= S. surattense), 3) S. torvum, 4) S. aethiopicum (= S. gilo), S. aethiopicum (= S. integrifolium), S. violaceum (= S. indicum) and S. violaceum (= S. sanitwongsei) and 5) S. mammosum. The restriction pattern with Alu I in each of the reciprocal hybrids between S. melongena 'Uttara' and S. aethiopicum (= S. integrifolium) was identical with that of seed parent. The present study demonstrated the availability of the PCR-RFLP analysis of cpDNA for assessing taxonomic relationships and identifying cytoplasmic parentage of interspecific hybrids in eggplant and related Solanum species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genomic gene encoding manganese peroxidase from a white-rot fungus Phanerochaete crassa WD1694

The gene encoding manganese peroxidase of a white-rot fungus Phanerochaete crassa WD1694 was cloned and sequenced. Four genomic clones were sequenced in which 3 clones were existed as alleles. The analysis of intron–exon structures divided the 4 clones into three subfamilies that corresponded to mnp2 and mnp3 of Phanerochaete chrysosporium, and a new subfamily possessing only five introns. The purified P. crassa WD1694 MnP consisted of 4 isozymes with same molecular weight, same N-terminal sequence, and different pI. N-terminal sequence of deduced protein of P. crassa mnpB3 gene was identical to those of 4 MnP isozymes; however, the other 3 mnp genes had different N-terminal sequence. The molecular weight of encoded mature protein of mnpB3 gene and purified MnP had a gap that could be difference between MnP proteins encoded by single gene. The results suggested that 4 MnP isozymes of P. crassa WD1694 arose from single gene.