The role of chlorophyllase in degreening canola (Brassica napus) seeds and its activation by sublethal freezing

Chlorophyllase mediates dephytylation of chlorophylls and pheophytins during seed degreening in canola ( Brassica napus cv. Westar). Degreening can be correlated with chlorophyllase activity in vitro, but it is difficult to demonstrate in vivo activity because of low levels of the dephytylated breakdown products during rapid degreening. If, however, degreening is inhibited by sublethal freezing, chlorophyllide and pheophorbide accumulation can be related to the action of chlorophyllase. Changes in the rate of in vitro dephytylation during degreening and the dramatic increase following freezing may indicate enzyme activation and de novo enzyme synthesis. Evidence from Western blots is presented in support of de novo synthesis. It is concluded that failure of the seed to degreen following sublethal freezing does not result from a reduction in chlorophyllase activity.     

A ratiometric fluorescent probe for assessing mitochondrial phospholipid peroxidation within living cells

Mitochondrial oxidative damage contributes to a wide range of pathologies, and lipid peroxidation of the mitochondrial inner membrane is a major component of this disruption. However, despite its importance, there are no methods to assess mitochondrial lipid peroxidation within cells specifically. To address this unmet need we have developed a ratiometric, fluorescent, mitochondria-targeted lipid peroxidation probe, MitoPerOx. This compound is derived from the C11-BODIPY(581/591) probe, which contains a boron dipyromethane difluoride (BODIPY) fluorophore conjugated via a dienyl link to a phenyl group. In response to lipid peroxidation the fluorescence emission maximum shifts from ～590 to ～520nm. To target this probe to the matrix-facing surface of the mitochondrial inner membrane we attached a triphenylphosphonium lipophilic cation, which leads to its selective uptake into mitochondria in cells, driven by the mitochondrial membrane potential. Here we report on the development and characterization of MitoPerOx. We found that MitoPerOx was taken up very rapidly into mitochondria within cells, where it responded to changes in mitochondrial lipid peroxidation that could be measured by fluorimetry, confocal microscopy, and epifluorescence live cell imaging. Importantly, the peroxidation-sensitive change in fluorescence at 520nm relative to that at 590nm enabled the use of the probe as a ratiometric fluorescent probe, greatly facilitating assessment of mitochondrial lipid peroxidation in cells.     

Tubes and tube-building in a lotic Chironomid (Diptera) community

The use of particles for the construction of tubes by a community of stream-dwelling midge (Chironomidae) larvae was investigated. It was shown that the particles present in the larval tubes occurred in the same proportions as those present on the surfaces of stones in the stream. Small particles were principally of detritus, medium-sized particles mainly of detritus and diatoms, and a a variety of types of particles, including filamentous algae and sand grains, occurred in the large size category. The absence of particle selection in the field was reflected in laboratory experiments. It is suggested that competition for suitable available space in which to build tubes and the quantity of building material are likely to be of more significance to the larval community in upland streams than particle size or type.     

Repeated structure and possible gene duplications in high potential iron protein and rubredoxin

Summary The three-dimensional structures of bacterial high potential iron protein (HIPIP) and rubredoxin have been searched for repeats to test whether these molecules evolved by independent tandem gene duplications. HIPIP has no structural repeats in spite of the observed repeated pattern in the amino acid sequence fromRhodopseudomonas gelatinosa. Rubredoxin fromClostridium pasteurianum has repeated hairpin loops of ten alpha-carbon atoms on both sides of the active centre iron-sulphur complex, which can be superposed within a root mean square deviation of 0.84 Å by rotating about a local pseudo-dyad axis. The structural repeat matches a weak repeat in the amino acid sequence. It is concluded that the sequence repeats in HIPIP are probably a coincidence but that rubredoxin may have evolved by gene duplication from a dimer of two primitive hairpin loops.