Xanthine oxidase and aldehyde oxidase: a simple procedure for the simultaneous purification from rat liver

Aldehyde oxidase (AO) and xanthine oxidase (XO) are cytosolic enzymes that have been involved in some pathological conditions and play an important role in the biotransformation of drugs and xenobiotics. The increasing interest in these enzymes demands for a simple and rapid procedure for their purification. This paper describes for the first time a method that allows simultaneous purification of both enzymes from the same batch of rat livers. It involves few steps, is reproducible and offers high enzyme yields with high specific activities. The rat liver homogenate was fractionated by heat denaturation and by ammonium sulphate precipitation to give a crude extract containing both enzymes. This extract was chromatographed on an Hydroxyapatite column that completely separated AO from XO. Further purification of XO by anion exchange chromatography on a Q-Sepharose Fast Flow column resulted in a highly purified (1200-fold) preparation, with a specific activity of 3.64 U/mg and with a 20% yield. AO was purified about 1000-fold at a yield of 15%, with a specific activity of 3.48 U/mg, by affinity chromatography on Benzamidine-Sepharose 6B. The purified enzymes gave single bands of approximately 300 kDa on a polyacrylamide gel gradient electrophoresis and displayed the characteristic absorption spectra of highly purified enzymes.     

Neo-clerodane diterpenoids from Croton schiedeanus

Two new neo-clerodane type furano diterpenoids were isolated from the aerial part of Croton schiedeanus, besides the clerodane diterpenes cis- and trans-dehydrocrotonin, previously isolated from other species of Croton. Structural elucidation was achieved on basis of extensive NMR experiments, including X-ray diffraction analysis and molecular mechanics calculations. The previously known flavonoids ayanin and quercetin-3,7-dimethyl ether were also obtained from the extract of this plant.     

Analysis of the argK-tox gene cluster in nontoxigenic strains of Pseudomonas syringae pv. phaseolicola

The analysis of 46 isolates obtained directly from different and distant common bean fields from the northwestern part of Spain revealed that they do not produce phaseolotoxin. The isolates were classified as race 5, and their analysis revealed that they do not carry the argK-tox gene cluster involved in the biosynthesis of the phaseolotoxin.     

Influence of maternal stress on uranium-induced developmental toxicity in rats

It has been demonstrated that uranium is an embryo/fetal toxicant when given orally or subcutaneously to pregnant mice. On the other hand, maternal stress has been shown to enhance the developmental toxicity of a number of metals. In this study, maternal toxicity and developmental effects of a concurrent exposure to uranyl acetate dihydrate (UAD) and restraint stress were evaluated in rats. Four groups of pregnant animals were given subcutaneous injections of UAD at 0.415 and 0.830 mg/kg/day on Days 6 to 15 of gestation. Animals in two of these groups were also subjected to restraint for 2 hr/day during the same gestational days. Control groups included restrained and unrestrained pregnant rats not exposed to UAD. Cesarean sections were performed on gestation Day 20, and the fetuses were weighed and examined for malformations and variations. Maternal toxicity and embryotoxicity were noted at 0.830 mg/kg/day of UAD, while fetotoxicity was evidenced at 0.415 and 0.830 mg/kg/day of UAD by significant reductions in fetal body weight and increases in the total number of skeletally affected fetuses. No teratogenic effects were noted in any group. Maternal restraint enhanced uranium-induced embryo/fetal toxicity only at 0.830 mg/kg/day, a dose that was also significantly toxic to the dams. As in previous studies with other metals, maternal stress enhances uranium-induced developmental toxicity at uranium doses that are highly toxic to the dams; however, at doses that are less acutely toxic the role of maternal stress would not be significant.     

Systemic induction of the biosynthesis of terpenic compounds in Digitalis lanata

A bacterial screening was carried out in the rhizosphere of two Digitalis species, D. thapsi and D. parviflora, both at the vegetative stage and at flowering. A total of 480 isolates were characterised at genus level, Bacillus being the dominant genera in all cases. Fifty percent of the Bacillus strains isolated from each species were analysed by PCR-RAPDs. At 85% similarity, 12 groups separated for D. thapsi and 18 for D. parviflora. One strain of each group was selected for biological assay on D. lanata, evaluating growth promotion and cardenolide content in leaves after inoculation performed in the root system. The plant parameters evaluated were leaf surface area, shoot and root dry weight and leaf number. Lanatoside C content was evaluated by HPLC. Only 17 strains caused significant increases in at least one of the parameters evaluated. The most striking result was that some strains promoted growth and increased cardenolide content at the same time. This effect was detected on leaves while inoculation was carried out on roots. Interestingly, these two parameters are not enhanced simultaneously under regular conditions in pot or in tissue cultures.     

Immunohistochemistry,ultrastructure and pathology of gills of Abramis brama from Lake Mondsee,Austria, infected with Ergasilus sieboldi (Copepoda)

Immunohistochemical, ultrastructural and pathological studies were carried out on the gills of bream Abramis brama (L.) from Lake Mondsee, Austria, that were naturally infected with Ergasilus sieboldi Nordmann, 1832. Of a total of 14 specimens of bream examined, the gills of 7 (50%) were parasitized with this copepod and the intensity of infection ranged from 1 to 23 crustaceans per host. Histopathological investigations of infected gill showed extensive tissue damage due to attachment and feeding of the crustacean. Parasites attached close to the base of filaments near the gill arch. Pressure exerted by the ectoparasite attached to the lateral margin of the gill filaments induced atrophy of the secondary lamellae. Tissue reactions included hyperplasia and mucous cell proliferation of the respiratory epithelium. Mucous cells displayed an intense immunohistochemical reactivity with the anti-nitric oxide synthase antibody. In parasitized primary and secondary lamellae, a high number of eosinophilic granular cells and rodlet cells were noticed. Rodlet cells represent an inflammatory cell type closely linked to other piscine inflammatory cells. Presence of a high number of inflammatory cells at the site of E. sieboldi attachment is related to intense host cellular reaction.