Molecular and functional characterization of a cDNA encoding fructan:fructan 6G-fructosyltransferase (6G-FFT)/fructan:fructan 1-fructosyltransferase (1-FFT) from perennial ryegrass (Lolium perenne L.)

Vol. 57 No. 11, 2006,     

Generation of Oxygen Radicals from Iron Complex of Orellanine, A Mushroom Nephrotoxin; Preliminary ESR and Spin-Trapping Stukes

Orellanine, [2,2'-bipyridine]-3,3',4,4'-tetrol-l,I'-dioxide, is the toxin responsible for the lethal nephrotoxicity of some Cortinarius mushrooms. Our present ESR and spin-trapping studies of the redox properties of the system of non-illuminated orellanine, ferrous iron and dioxygen contribute to understanding the molecular mechanism of its toxicity. UV-visible spectrophoto-metry, cyclic voltammetry and ESR in frozen medium showed the formation of a wine-red tris complex, Fe(III)Or₃. This ferric complex is easily reducible (E_P =-565 mV vs Ag/AgCl/3M KCl at pH7), involving a one-electron reversible process. Spin-trapping using DMPO is employed to detect the generation of super-oxide anion and hydroxyl radicals. The instantaneous one-electron oxidation of ferrous ions in the presence of the toxin under air is concomitant with dioxygen consumption as supported by dioxygen consumption. GSH involves the toxin and ferrous ions under air in a redox cycling process resulting in the production of glutathionyl and oxygen free radicals, observed for the first time with an iron complex of a mushroom toxin. In most cases, EDTA is not able to prevent the Fe(III)Or₃ and radical formation. The ortho-dihydroxylated groups borne by the di-N-oxidized bipyridine structure and not the bipyridine structure itself, are responsible for the formation of a stable ferric complex at pH 7, as they are for the generation of an apparently stable ortho-semiquinone anion radical. These one-electron mechanisms may play a major role in some of the known toxic effects of orellanine.     

Evaluation of different methods to assess the side-effects of pesticides on Phytoseiulus persimilis A.-H.

Two methods recommended by the International Organization for Biological and Integrated Control of Noxious Animals and Plants (IOBC) to test the side-effect of pesticide residues on Phytoseiulus persimilis in the laboratory were evaluated. The test on detached leaves resulted in an unacceptably high percentage of escaping individuals. This was not the case when the tests were performed in ventilated glass cages. To validate the results obtained in the laboratory additional experiments were performed on intact plants in the greenhouse. Results obtained on detached leaves underestimate effects found in the greenhouse, whereas effects measured in the glass cages are an overestimation. Besides the effect of residue on juvenile survival and adult reproduction, ovicidal effects and the effect of direct contact for adults and eggs were also taken into account in the evaluation. All tests were performed with endosulfan. It is concluded that the use of this compound at the recommended field concentration is not compatible with the use of P. persimilis.     

Diel thermoregulation of the crawfish Procambarus Clarkii (crustacea, cambaridae)

1. 1. In a diel cycle Procambarus clarkii has two preferred temperatures: 24.0 ± 0.15 SEM °C during the day and 26.7 ± 0.13 SEM °C at night.

2. 2. The preferred temperatures are independent from the weight of the organisms.

3. 3. In the photophase the animals are dispersed, in the scotophase they congregate.

4. 4. The crawfish seem to feed during the thermal interphases.

5. 5. Animals in a constantly dark condition maintain a diel preferendum of temperature.

Arginine modifiers as energy transfer inhibitors in photophosphorylation

Photophosphorylation by spinach chloroplasts is inhibited after they have been incubated in the dark with either phenylglyoxal or butanedione. Inhibition by phenylglyoxal is strongest when N-ethylmorpholine is the buffer used during the incubation; that by butanedione requires the presence of borate as buffer. The inhibitions are not reversed by simply washing out the inhibitor, suggesting that a covalent modification of one or more arginine residues is responsible. This is supported by the reversibility of the butanedione inhibition if both the inhibitor and borate buffer are removed. ATPase of the chloroplasts, and of extracted protein, is inhibited, whether activated by trypsin or by heating. This indicates that arginine residues of the coupling factor are the probable major site(s) for attack by these modifiers, leading to the observed inhibitions.     

A new spin-labelled analogue of nicotinamide adenine dinucleotide

The preparation of a spin-labelled analogue of nicotinamide adenine dinucleotide, 3-(4′,4′,5′,5′-tetramethyl-3′-oxide-1′-oxyl-2′-imidazolinyl) pyridine adenine dinucleotide, is descrebed. This compound was obtained by treatment of 3-carboxaldehyde pyridine adenine dinucleotide with 2,3-dimethyl-2,3-dihydroxylaminobutane followed by oxidation with lead dioxide. The interpretation of the particular electron spin resonance spectra of this nitronylnitroxide (five lines) in terms of the rotational correlation time of the radical is shown to be possible. The high stability of this compound makes its use in NAD⁺-dependent biological systems feasible.     

Kinetics of partly diffusion-controlled reactions

Spectrofluorimetric methods allowing an estimation of the “microviscosity” (or the microfluidity) of synthetic and natural membranes may be used if the emisive phenomenon is dependent upon the cohesion of its local environment. Of the different methods that may be proposed, the study of the reactions between electronically excited molecules A^*, which emit fluorescence, and B quenchers embedded in membranes, the rate constant of which is partly controlled by diffusion, are expected to inform about the values of diffusion coefficients of quenchers and therefore the “microviscosity” of the environment. Information may be improved when the product of the reaction between A^* and B is itself emissive (excimers, exciplexes). We propose here that a kinetic model may apply this type of reaction when a process of static quenching occurs. The use of this kinetic model in studies carried out in this area should lead to a more accurate determination of the diffusion coefficients of A^* and B and therefore of the “microviscosity.”     

Multiple forms of oviduct progesterone receptors analyzed by ion exchange filtration and gel electrophoresis.

Resolution of the multiple forms of steroid receptors in small samples has been improved by two new techniques: preparative ion exchange filtration and electrophoresis in highly cross-linked polyacrylamide gels of varied concentration. These techniques were used in conjunction with protamine precipitation, gel filtration, and density gradient centrifugation to separate five forms of the progesterone receptor of chick oviduct cytosol. These complexes, numbered I to V in order of elution from agarose gel columns, have been characterized with respect to apparent molecular weight, shape, and relative net charge. Form I, which is eluted in the void volume after gel filtration of cytosol in hypotonic media, is heterodisperse with respect to sedimentation coefficient and electrophoretic mobility (Rf). Form I is converted to form III by KC1. Form II has the highest axial ratio and the highest Rf at pH 10.2. This 4.2S complex can be extracted from DEAE filters, but not from protamine-precipitated cytosol, by 0.3 to 0.5 M KC1. Form III is slightly smaller (3.9S) and less asymmetric than form II. It is relased from DEAE filters and protamine-precipitated cytosol by 0.15 M KC1 and displays increased Rf upon purification. Forms II and III correspond to the B and A components described by W. T. Schrader and B. W. O'Malley ((1972), J. Biol. Chem 247, 51). Form IV may result from the proteolytic cleavage of forms II and/or III. Form V is a globular polypeptide obtained in the presence of certain divalent cations. This complex has been named the "mero-receptor" since it is the smallest part or fragment of the receptor that contains the steroid-binding site.