Synthesis and properties of azidonitrophenyl pyrophosphate, a photoaffinity probe of the nucleotide binding sites of mitochondrial F1-ATPase

4-Azido-2-nitrophenyl pyrophosphate (azido-PPi) labeled with 32P in the alpha position was prepared and used to photolabel beef heart mitochondrial F1. Azido-PPi was hydrolyzed by yeast inorganic pyrophosphatase, but not by mitochondrial F1-ATPase. Incubation of F1 with [alpha-32P]azido-PPi in the dark under conditions of saturation resulted in the binding of the photoprobe to three sites, two of which exhibited a high affinity (Kd = 2 microM), the third one having a lower affinity (Kd = 300 microM). Mg2+ was required for binding. As with PPi [Issartel et al. (1987) J. Biol. Chem. 262, 13538-13544], the binding of 3 mol of azido-PPi/mol of F1 resulted in the release of one tightly bound nucleotide. ADP, AMP-PNP, and PPi competed with azido-PPi for binding to F1, but Pi and the phosphate analogue azidonitrophenyl phosphate did not. The binding of [32P]Pi to F1 was enhanced at low concentrations of azido-PPi, as it was in the presence of low concentrations of PPi. Sulfite, which is thought to bind to an anion-binding site on F1, inhibited competitively the binding of both ADP and azido-PPi, suggesting that the postulated anion-binding site of F1 is related to the exchangeable nucleotide-binding sites. Upon photoirradiation of F1 in the presence of [alpha-32P]azido-PPi, the photoprobe became covalently bound with concomitant inactivation of F1. The plots relating the inactivation of F1 to the covalent binding of the probe were rectilinear up to 50% inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of four herbicide-resistant mutants of Rhodopseudomonas viridis by genetic analysis, electron paramagnetic resonance, and optical spectroscopy

Herbicides of the triazine class block electron transfer in the photosynthetic reaction centers of purple bacteria and PSII of higher plants. They are thought to act by competing with one of the electron acceptors, the secondary quinone, QB, for its binding site. Several mutants of the purple bacterium Rhodopseudomonas viridis resistant to terbutryn [2-(methylthio)-4-(ethylamino)-6-(tert-butylamino)-s-triazine] have been isolated by their ability to grow photosynthetically in the presence of the herbicide. Sequence analysis of the genes coding for the L and M subunits of the reaction center showed that four different mutants were obtained, two of them being double mutated: T1 (SerL223----Ala and ArgL217----His), T3 (PheL216----Ser and ValM263----Phe), T4 (TyrL222----Phe), and T6 (PheL216----Ser). The residues L223 and L216 are involved in binding of QB, whereas L217 and L222 are not. M263 is part of the binding pocket of the primary quinone, QA. The affinity of the reaction centers for terbutryn and the electron transfer inhibitor o-phenanthroline, determined via the biphasic charge recombination after one flash, is decreased for all mutants. The affinity for ubiquinone 9 is also decreased, except in T1. Characterization by EPR spectroscopy showed that the QB.-Fe2+ signal of T4, having a g = 1.93 peak, is different from the signals obtained with the wild type and the other mutants but very similar to those of Rhodospirillum rubrum and PSII. The results obtained by the combination of these different techniques are discussed with respect to the three-dimensional structure of the wild type and the mode of binding of ubiquinone, terbutryn, and o-phenanthroline as determined by X-ray structure analysis.     

Biofeedback control of migraine headaches: A comparison of two approaches

In order to assess the relative effectiveness of finger warming and temporal blood volume pulse reduction biofeedback in the treatment of migraine, 22 female migraine patients were assigned to one of three experimental conditions: temporal artery constriction feedback, finger temperature feedback, or waiting list. Biofeedback training consisted of 12 sessions over a 6-week period. All patients completed 5 weeks of daily self-monitoring of headache activity (frequency, duration, and intensity) and medication before and after treatment. Treatment credibility was assessed at the end of Sessions 1, 6, and 12. Results showed that temporal constriction and finger temperature biofeedback were equally effective in controlling migraine headaches and produced greater benefits than the waiting list condition. Power analyses indicated that very large sample sizes would have been required to detect any significant differences between the two treatment groups. No significant relationships were found between levels of therapeutic gains and levels of thermal or blood volume pulse self-regulation skills. Likewise, treatment outcome was not found to be related to treatment credibility. Further analyses revealed that changes in headache activity and medication were associated with changes in vasomotor variability. Because blood volume pulse variability was not significantly affected by biofeedback training, questions about its role in the therapeutic mechanism are raised.This research was supported in part by grants from the Quebec Ministry of Education and the Quebec Ministery of Social Affairs to the first author, and an award from the Medical Research Council of Canada to the second author. The authors are indebted to Drs. Frank Andrasik, Howard Barbaree, Edward Blanchard, Martin Ford, and Patrick McGrath, as well as to two anonymous reviewers, for their helpful comments on an earlier draft of this paper.     

Characterization of a new class of circular DNA molecules in yeast

This paper describes the isolation of a pure population of covalently closed circular twisted DNA molecules from yeast. These molecules are homogeneous in size, that is consist of monomers of 2.2μ and of multiple length oligomers of n x 2.2μ. While no data rule out the mitochondrial origin of this DNA, its actual intracellular localization remains unknown; it displays the same buoyant density as the main nuclear DNA and therefore is not the heavy nuclear satellite DNA (γ-DNA described by Moustacchi and Williamson (1966)); although circular molecules represent only 1 to 5 % of the total DNA, they can be prepared in sizable and reproducible amounts by a method based on the use of mechanical disruption of yeast cells rather than lysis by snail gut juice.     

Cellular Studies of X-Ray Induced Inhibition of Lens Regeneration

Whole-body X-irradiation of adult newts 0 to 3 days after lentectomy inhibits transformation of the dorsal iris epithelium into a lens in all cases. The first question raised was whether irradiation affects infiltration of the iris area by macrophages, and the phagocytic activities of these cell types in the iris epithelium (prominent phenomena in this system). The number of macrophages infiltrating into the iris epithelium, and their phagocytic activities (indicated by uptake of melanosomes) were not affected by irradiation under those conditions. The second group of experiments concerns the possible effects of irradiation on DNA replication of iris epithelial cells, which become transformed into lens cells in the non-irradiated system. Autoradiographic studies of iris epithelial cells in vivo revealed a significant suppressive effect of irradiation on the frequencies of cells incorporating ³H-thymidine 7 and 14 days after lentectomy. When autoradiography was applied to the primary pure culture of iris epithelial cells at different time intervals after the start of culture and irradiation in vitro , significant and persistent reduction of cell labelling due to irradiation, was demonstrated. Multiplication of spread cells in the iris epithelial culture was strongly and persistently inhibited throughout a period of 2 months. Inhibition of cell labelling and of cell multiplication was always accompanied by reduction in the extent of de-pigmentation of iris epithelial cells. De-pigmentation is one of the requirements for the cells become transformed into lens cells. The possible mechanism of radiation-induced inhibition of lens regeneration is discussed.     

Modelling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis

Alignment of the 87 available sequences of group I self-splicing introns reveals numerous instances of covariation between distant sites. Some of these covariations cannot be ascribed to historical coincidences or the known secondary structure of group I introns, and are, therefore, best explained as reflecting tertiary contacts. With the help of stereochemical modelling, we have taken advantage of these novel interactions to derive a three-dimensional model of the conserved core of group I introns. Two noteworthy features of that model are its extreme compactness and the fact that all of the most evolutionarily conserved residues happen to converge around the two helices that constitute the substrate of the core ribozyme and the site that binds the guanosine cofactor necessary for self-splicing. Specific functional implications are discussed, both with regard to the way the substrate helices are recognized by the core and possible rearrangements of the introns during the self-splicing process. Concerning potential long-range interactions, emphasis is put on the possible recognition of two consecutive purines in the minor groove of a helix by a GAAA or related terminal loop.