Interaction between cytochrome c and cytochrome c peroxidase: excited-state reactions of zinc- and tin-substituted derivatives

The effect of cytochrome c peroxidase (CCP) and apoCCP on the fluorescence and phosphorescence of Zn and Sn cytochrome c (cyt c) and the effect of cyt c on the fluorescence and phosphorescence of Zn CCP were examined. We found the following: The fluorescence yields of Zn and Sn cyt c were quenched by about 20% by CCP, consistent with energy transfer between the two chromophores with a separation of about 1.8 nm. The phosphorescence spectrum of Zn cyt c (but not Sn cyt c) shifts by 20 nm to the blue upon complexation with either CCP or apoCCP; at the same time the phosphorescence lifetime of Zn cyt c decreases from 12 +/- 2 to 6 ms with apoCCP addition. Zn CCP phosphorescence decay increases from 8.3 to 9.1 ms upon addition of poly(L-lysine) used to mimic cyt c. It is concluded from these results that binding of the redox partner or an analogue to Zn CCP and Zn cyt c results in a conformational change. The respective phosphorescence lifetimes of Zn and Sn cyt c were 13 and 3 ms in the absence of CCP and 1.6 and 1.1 ms in the presence of CCP; this corresponds to a quenching rate due to CCP of 519 and 570 s-1, for Zn and Sn cyt c, respectively. The phosphorescence of Zn CCP is also affected by native cyt c but is dramatically less than the complementary pair; the quenching rate constant is 17 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium/plant cell interaction

Summary We investigated whether the efficiency of transformation of plant cells by Agrobacterium tumefaciens during cocultivation is limited by the properties of the plant cells or by the infecting bacteria.Therefore, tobacco protoplasts were infected by cocultivation with two different agrobacteria strains carrying Ti plasmids with distinguishable T-DNAs. These T-DNAs cotransform plant cells at a frequency equal to the product of their independent transformation frequencies, which indicates that all plant cells are equally competent. On the other hand, when these T-DNAs are located on the same Ti plasmid vector within one bacterial strain, the cotransformation frequency is significantly higher than the product of the single transformation frequencies. We interpret these results to indicate that transformation is limited more by the establishment of effective bacteria/plant cell interaction than by (i) the process of DNA integration and (ii) by the number of plant cells capable of being transformed by Agrobacterium. We found that most plant cells are transformed by only one or a few agrobacteria. Analysis of the number of T-DNA copies in these clonally transformed lines indicates amplification of the original, infecting T-region copy.     

Structural characterization of cytochrome c peroxidase by resonance Raman scattering

Resonance Raman scattering studies are reported on freshly prepared and aged ferric, ligand-free ferrous, and CO-bound ferrous cytochrome c peroxidase. The ferric form of the fresh enzyme has a heme which is penta-coordinate high spin, independent of buffer over the pH range 4.3-7, as determined by well established Raman marker lines. The aged enzyme displays a mixture of spin and coordination states, but it can be stabilized in the penta-coordinate high spin form in the presence of phosphate. These results can be accounted for by considering the size of the channel (6 A wide, 11 A long) between the distal side of the heme and the outer surface of the protein. A phosphate ion may be accommodated in this channel resulting in the stabilization of the distal heme pocket. The ferrous cytochrome c peroxidase in both the ligand-free and CO-bound states has an acidic and an alkaline form. The acidic form has the characteristic spectral features of peroxidases: a high frequency iron-histidine stretching mode (248 cm-1), a high frequency Fe-CO stretching mode (537 cm-1), and a low frequency C-O stretching mode (1922 cm-1). At alkaline pH these frequencies become similar to those of hemoglobin and myoglobin, with the corresponding modes located at 227, 510, and 1948 cm-1, respectively. We attribute the acid/alkaline transition in the ferrous forms of cytochrome c peroxidase to a rearrangement mainly of the proximal side of the heme, culminating in a change of steric interactions between the proximal histidine and the heme or of the hydrogen bonding network involving the proximal histidine. The new data presented here reconcile many inconsistencies reported in the past.     

New Zealand machair vegetation

Abstract. Machair vegetation is reported for the first time from New Zealand. The habitat is similar to that of British machairs in climate, topography and generally in soil. pH and CaCO₃ content are much lower through most of the sequence, though this difference may partly reflect the greater disturbance of British machair. Sea machair is present, predominantly comprising native species. This grades into machair proper, which contains many species found also in British machair. The machair includes Ammophila-occupied hillocks, a feature typical of British machair. Machair marsh is also present.     

Lawns have vertical stratification

Abstract. Vertical stratification was examined in a closely-mown lawn, at both the beginning and end of the mowing/regrowth cycle. Two treatments were examined: Undisturbed, which had been maintained as lawn for 30 yr, and Mechanically Perturbed, on which succession was occurring, after complete removal of vegetation 9.5 months previously. There was significant vertical stratification at both stages of the mowing/regrowth cycle, i.e. even immediately after mowing to a height of 3 cm, and in both treatments. There was general consistency of the vertical role of species during the cycle and between treatments. Relative vertical positions were not closely correlated with absolute leaf heights. However, in no case was the vertical position of a pair of species significantly reversed during the cycle or between treatments. No mutually cyclic vertical relations between species were seen. It is concluded that vertical stratification occurs in even the shortest communities, it starts to develop quite early in succession, and it is dependent largely on the intrinsic properties of the species, their height and (at least in lawns) their recovery from defoliation.     

Responses of barley,pea, and maize to inoculation with different vesicular-arbuscular mycorrhizal fungi in irradiated soil

Summary The efficiency of different VAM fungi was investigated by inoculating barley, pea, and maize with different VAM fungi in irradiated soil in pots buried in the field. VAM frequency, growth and nutrient uptake were measured. In barleyGlomus epigaeus (CA) andG. macrocarpus (CA) were the most efficient out of 11 tested species and increased yield of grain by 24% and 21%, though they were not significant according to oneway analysis of variance. In pea, yield of grain was significantly increased from 46% to 104% (mean=68%) by 7 out of 10 tested species and by 105% by application of P fertilizer. The most efficient species wereG. epigaeus (CA),G. mosseae (GB), andG. etunicatus (CA). In maizeG. mosseae (GB) andG. caledonius (DK) increased total yield significantly by 59% and 47% in one experiment and in another experiment yield of cob was increased by 68% byG. mosseae (GB), 72% byG. caledonius (DK), and by 153% by application of P fertilizer. This experiment demonstrated that responsiveness to inoculation by VAM fungi differed among plant species, and that efficiency of different VAM fungi differed.     

The occrrence of vesicular-arbuscular mycorrhiza in barley and wheat grown in some Danish soils with different fertilizer treatments

Summary Soil samples, roots and shoots were collected from barley crops at three locations which had received different combinations of N and P for 10 years, and from long-term fertilizer experiments on barley in a sandy soil and on barley and wheat in a loamy soil.Soils were analysed for available P by an anion exchange resin procedure, roots were examined for intensity of VAM infection, and shoots wee analysed for N and P.Vesicualr-arbuscular mycorrhizal infection was found at all locations. It was most abundant at the three locations with least soil-P and lightest at the two locations high in soil-P. Within loocations an inverse relation was found between soil-P level and intensity of infection. Infection was also intensity. by increasing N-fertilizer. Spore counts from selected samples correlated well with infection intensity. Shoot-P did not differ significantly between treatments in spite of significant differences in soil-P. This points to the significance of vesicular-arbuscular mycorrhiza: a lower soil-P level accompanied by a higher infection intensity seem to counterbalance each other to a certain extent.     

Polypeptide synthesis by mitoplasts isolated from mouse liver

Polypeptide synthesis by mouse liver mitochondria was studied by incubating purified mitoplasts (mitochondria treated with digitonin) with [³⁵S]methionine. The products were separated either by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, or by isoelectric focusing, followed by SDS polyacrylamide gel electrophoresis. At least 14 distinct bands with molecular weights (mol. wt) ranging from about 8 000 to about 70 000 were found upon radioautography of the gels. When the samples were incubated in the presence of chloramphenicol, only a single weak band was found, whereas the protein pattern was unaffected by the presence of cycloheximide in the medium. The newly synthesized proteins were all acidic and evidence was obtained that they were hydrophobic in nature. Virtually all the labelled polypeptides were present in the membrane fraction, whereas the matrix showed little radioactivity. The data indicate that the proteins synthesized by mammalian mitochondria, like those in yeast, are components of the inner mitochondrial membrane. One protein of mol. wt 22 000 D was detected in the incubation medium. Since more of this component was present in the medium than in the pelleted mitoplasts and since this protein was not found in the matrix fraction of sonicated mitoplasts, it is believed that it had been excreted from the inner mitochondrial membrane. The finding that the number of proteins synthesized in mitoplasts isolated from mouse liver is considerably higher than that synthesized in yeast mitochondria reflects a most efficient utilization of the mammalian mitochondrial genome.     

MRX protects fork integrity at protein–DNA barriers,and its absence causes checkpoint activation dependent on chromatin context

To address how eukaryotic replication forks respond to fork stalling caused by strong non-covalent protein–DNA barriers, we engineered the controllable Fob-block system in Saccharomyces cerevisiae. This system allows us to strongly induce and control replication fork barriers (RFB) at their natural location within the rDNA. We discover a pivotal role for the MRX (Mre11, Rad50, Xrs2) complex for fork integrity at RFBs, which differs from its acknowledged function in double-strand break processing. Consequently, in the absence of the MRX complex, single-stranded DNA (ssDNA) accumulates at the rDNA. Based on this, we propose a model where the MRX complex specifically protects stalled forks at protein–DNA barriers, and its absence leads to processing resulting in ssDNA. To our surprise, this ssDNA does not trigger a checkpoint response. Intriguingly, however, placing RFBs ectopically on chromosome VI provokes a strong Rad53 checkpoint activation in the absence of Mre11. We demonstrate that proper checkpoint signalling within the rDNA is restored on deletion of SIR2. This suggests the surprising and novel concept that chromatin is an important player in checkpoint signalling.