Mode of Action Studies on a Chiral Diphenyl Ether Peroxidizing Herbicide: Correlation between Differential Inhibition of Protoporphyrinogen IX Oxidase Activity and Induction of Tetrapyrrole Accumulation by the Enantiomers

The nitrodiphenyl ether herbicide 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitroacetophenone oxime-O-(acetic acid, methyl ester) (DPEI) induced an abnormal accumulation of protoporphyrin IX in darkness in the green alga Chlamydomonas reinhardtii, as determined by high-performance liquid chromatography and spectrofluorimetry. It also inhibited the increase in cell density of the alga in light-grown cultures with an I₅₀ (concentration required to decrease cell density increase to 50% of the noninhibited control value) of 0.16 μm. The relative ability of four peroxidizing diphenyl ether herbicides to cause tetrapyrrole accumulation in C. reinhardtii correlated qualitatively with their ability to inhibit the increase in cell density in light-grown cultures. The purified S(−) enantiomer of the optically active phthalide DPE 5-[2-chloro-4-(trifluoromethyl)phenoxy]-3-methylphthalide (DPEIII), which has greater herbicidal activity than the R(+) isomer, induces a 4- to 5-fold greater tetrapyrrole accumulation than the R(+) isomer. The I₅₀ for inhibition of increase in cell density in light-grown cultures of C. reinhardtii by the S(−) isomer (0.019 μm) is less than 25% of that for the R(+) isomer. DPEIII inhibits protoporphyrinogen IX oxidase activity in pea (Pisum sativum) etioplast lysates, with the S(−) enantiomer showing considerably greater potency than the R(+) isomer and the racemic mixture showing a potency intermediate between the two. The results indicate that the site at which DPEs inhibit protoporphyrinogen IX oxidase shows chiral discrimination and provide further evidence for the link between inhibition of this enzyme, protoporphyrin IX accumulation, and the phytotoxicity of DPE herbicides.     

Cytopathology of Anthers and Pollen From Lettuce Plants Infected by Lettuce Mosaic Virus

Thin sections of mature anthers and pollen grains from three lettuce (Lactuca sativa) plants infected with lettuce mosaic potyvirus (LMV) were studied by immunogold labelling. Labelled LMV particles were present externally on the exine of pollen grains from all plants, but were observed internally in the pollen grains from only one plant. Within mature pollen grains the virus particles were associated with the cytoplasmic bundle inclusions typical of infection by potyviruses. The tapetal plasmodium and the epidermal and endothecial layers of mature anthers from all infected plants also contained labelled virus particles, together with pinwheel and bundle inclusions.     

Determination of d-amphetamine in biological samples using high-performance liquid chromatography after precolumn derivatization with o-phthaldialdehyde and 3-mercaptopropionic acid

An HPLC method is described for the determination of amphetamine using fluorometric detection after derivatization with o-phthaldialdehyde and 3-mercaptopropionic acid. This procedure is more sensitive (detection limit 370 fmol in microdialysate buffer standards, 1.5 pmol in extracted plasma and tissue samples) than most of the previous methods described for the determination of amphetamine with HPLC-fluorescence detection. Due to the stability of the derivative it is also suitable for autosampling after manual derivatization. Investigators currently using o-phthaldialdehyde derivatization and fluorometric detection for amino acid determination should be able to rapidly implement this method.     

Membrane-bound apolipoprotein B is exposed at the cytosolic surface of liver microsomes.

We have used a competitive enzyme-linked immunoassay with a panel of monoclonal antibodies to probe the topography of the membrane-bound form of apolipoprotein B (apo B) in rabbit microsomes. All epitopes investigated were found to be expressed at the cytosolic side of the microsomal membrane under conditions in which the vesicles remained sealed. These results indicate that the membrane-associated form of apolipoprotein B is either at the cytosolic side of the endoplasmic reticulum membrane or integrated into the membrane. From this site apo B may be translocated to the lumen for assembly into VLDL or may be degraded.     

Biomarker responses in river otters experimentally exposed to oil contamination

Investigations in Prince William Sound (Alaska, USA) following the Exxon Valdez oil spill (EVOS) revealed that river otters (Lontra canadensis) on oiled shores had lower body mass and elevated values of biomarkers, than did otters living on nonoiled shores. In addition, otters from oiled areas selected different habitats, had larger home ranges, and less diverse diets than animals living in nonoiled areas. These differences between river otters from oiled shores and those from nonoiled areas strongly suggested that oil contamination had an effect on physiological and behavioral responses of otters. In this study, we explored the effects of crude oil contamination on river otters experimentally. We hypothesized that exposure to oil would result in elevated values of biomarkers, indicating induced physiological stress. Fifteen wild-caught male river otters were exposed to two levels of weathered crude oil (i.e., control, 5 ppm/day/kg body mass, and 50 ppm/day/kg body mass) under controlled conditions in captivity at the Alaska Sealife Center in Seward (Alaska, USA). Responses of captive river otters to oil ingestion provided mixed results in relation to our hypotheses. Although hemoglobin (Hb, and associated red blood cells) and white blood cells, and possibly interleukin-6 immunoreactive responded in the expected manner, other parameters did not. Aspartate aminotransferase, alanine aminotransferase, and haptoglobin (Hp), did not increase in response to oiling or decreased during rehabilitation. Conversely, principle-component analysis identified values of alkaline phosphatase as responding to oil ingestion in river otters. Our results suggested that opposing processes were concurring in the oiled otters. Elevated production of Hp in response to tissue damage by hydrocarbons likely occurred at the same time with increased removal of Hp-Hb complex from the serum, producing an undetermined pattern in the secretion of Hp. Thus, the use of individual biomarkers as indicators of exposure to pollutants may lead to erroneous conclusions because interactions in vivo can be complicated and act in opposite directions. Additionally, the biomarkers used in investigating effects of oiling on live animals usually are related to the heme molecule. Because of the opposing processes that may occur within an animal, data from a suite of heme-related biomarkers may produce results that are difficult to interpret. Therefore, we advocate the exploration and development of other biomarkers that will be independent from the heme cycle and provide additional information to the effect of oiling on live mammals.     

Resolved difference spectra of redox centers involved in photosynthetic electron flow in Rhodopseudomonas capsulata and rhodopseudomonas sphaeroides

1. In Rhodopseudomonas sphaeroides the Q_x absorption band of the reaction center bacteriochlorophyll dimer which bleaches on photo-oxidation is both blue-shifted and has an increased extinction coefficient on solubilisation of the chromatophore membrane with lauryldimethylamine-N-oxide. These effects may be attributable in part to the particle flattening effect.2. The difference spectrum of photo-oxidisable c type cytochrome in the chromatophore was found to have a slightly variable peak position in the α-band (λ_max at 551–551.25 nm); this position was always red-shifted in comparison to that of isolated cytochrome c₂ (λ_max at 549.5 ± 0.5 nm). The shift in wavelength maximum was not due to association with the reaction center protein. A possible heterogeneity in the c-type cytochromes of chromatophores is discussed.3. Flash-induced difference spectra attributed to cytochrome b were resolved at several different redox potentials and in the presence and absence of antimycin. Under most conditions, one major component, cytochrome b₅₀ appeared to be involved. However, in some circumstances, reduction of a component with the spectral characteristics of cytochrome b_?90 was observed.4. Difference spectra attributed to (BChl)₂, Q^?_II, c type cytochrome and cytochrome b₅₀ were resolved in the Soret region for Rhodopseudomonas capsulata.5. A computer-linked kinetic spectrophotometer for obtaining automatically the difference spectra of components functioning in photosynthetic electron transfer chains is described. The system incorporates a novel method for automatically adjusting and holding the photomultiplier supply voltage.     

Function of the iron-sulfur protein of the cytochrome b-c1 segment in electron transfer reactions of the mitochondrial respiratory chain

Resolution and reconstitution has been used to examine the involvement of the iron-sulfur protein of the cytochrome b-c1 segment in electron transfer reactions in this region of the mitochondrial respiratory chain. The iron-sulfur protein is required for electron transfer from succinate and from ubiquinol to cytochrome c1. It is not required for reduction of cytochrome b under these conditions, but it is required for oxidation of cytochrome b by cytochrome c plus cytochrome c oxidase. Removal of the iron-sulfur protein from the b-c1 complex prevents reduction of both cytochromes b and c1 by succinate or ubiquinol if antimycin is added to the depleted complex. As increasing amounts of iron-sulfur protein are reconstituted to the depleted complex, the amounts of cytochromes b and c1 reduced by succinate in the presence of antimycin increase and closely parallel the amounts of ubiquinol-cytochrome c reductase activity restored to the reconstituted complex, measured before addition of antimycin. The function of the iron-sulfur protein in these oxidation-reduction reactions is consistent with a cyclic pathway of electron transfer through the cytochrome b-c1 complex, in which the iron-sulfur protein functions as a ubiquinol-cytochrome c1/ubisemiquinone-cytochrome b oxidoreductase.     

Photosynthetic membrane development in Rhodopseudomonas sphaeroides. Spectral and kinetic characterization of redox components of light-driven electron flow in apparent photosynthetic membrane growth initiation sites

The kinetics of light-driven electron flow and the nature of redox centers at apparent photosynthetic membrane growth initiation sites in Rhodopseudomans sphaeroides were compared to those of intracytoplasmic photosynthetic membranes. In sucrose gradients, these membrane growth sites sediment more slowly than intracytoplasmic membrane-derived chromatophores and form an upper pigmented band. Cytochromes c1, c2, b561, and b566 were demonstrated in the upper fraction by redox potentiometry; c-type cytochromes were also detected electrophoretically. Signals characteristic of light-induced reaction center bacteriochlorophyll triplet and photooxidized reaction center bacteriochlorophyll dimer states were observed by EPR spectroscopy but the Rieske iron-sulfur signal of the ubiquinol-cytochrome c2 oxidoreductase was present at a 3-fold reduced level on a reaction center basis in comparison to chromatophores. Flash-induced absorbance measurements of the upper pigmented fraction demonstrated reaction center primary and secondary semiquinone anion acceptor signals, but cytochrome b561 photoreduction and cytochrome c1/c2 reactions occurred at slow rates. This fraction was enriched approximately 2- and 4-fold in total b- and c-type cytochromes, respectively, per reaction center over chromatophores, but photoreducible b-type cytochrome was lower. Measurements of respiratory activity indicated a 1.6-fold higher level of succinate-cytochrome c oxidoreductase/reaction center than in chromatophores, but the apparent turnover rates in both preparations were low. Overall, the results suggest that complete cycles of rapid, light-driven electron flow do not occur merely by introduction of newly synthesized reaction centers into respiratory membrane, but that subsequent synthesis and assembly of appropriate components of the ubiquinol-cytochrome c2 oxidoreductase is required.